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Sample records for estradiol inhibits chondrogenic

  1. IL-17 inhibits chondrogenic differentiation of human mesenchymal stem cells.

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    Masahiro Kondo

    Full Text Available OBJECTIVE: Mesenchymal stem cells (MSCs can differentiate into cells of mesenchymal lineages, such as osteoblasts and chondrocytes. Here we investigated the effects of IL-17, a key cytokine in chronic inflammation, on chondrogenic differentiation of human MSCs. METHODS: Human bone marrow MSCs were pellet cultured in chondrogenic induction medium containing TGF-β3. Chondrogenic differentiation was detected by cartilage matrix accumulation and chondrogenic marker gene expression. RESULTS: Over-expression of cartilage matrix and chondrogenic marker genes was noted in chondrogenic cultures, but was inhibited by IL-17 in a dose-dependent manner. Expression and phosphorylation of SOX9, the master transcription factor for chondrogenesis, were induced within 2 days and phosphorylated SOX9 was stably maintained until day 21. IL-17 did not alter total SOX9 expression, but significantly suppressed SOX9 phosphorylation in a dose-dependent manner. At day 7, IL-17 also suppressed the activity of cAMP-dependent protein kinase A (PKA, which is known to phosphorylate SOX9. H89, a selective PKA inhibitor, also suppressed SOX9 phosphorylation, expression of chondrogenic markers and cartilage matrix, and also decreased chondrogenesis. CONCLUSIONS: IL-17 inhibited chondrogenesis of human MSCs through the suppression of PKA activity and SOX9 phosphorylation. These results suggest that chondrogenic differentiation of MSCs can be inhibited by a mechanism triggered by IL-17 under chronic inflammation.

  2. Norepinephrine inhibition of mesenchymal stem cell and chondrogenic progenitor cell chondrogenesis and acceleration of chondrogenic hypertrophy.

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    Jenei-Lanzl, Zsuzsa; Grässel, Susanne; Pongratz, Georg; Kees, Frieder; Miosge, Nicolai; Angele, Peter; Straub, Rainer H

    2014-09-01

    Mesenchymal progenitor cell chondrogenesis is the biologic platform for the generation or regeneration of cartilage, but the external influence of the sympathetic nervous system on this process is not yet known. Sympathetic nerve fibers are present in articular tissue, and the sympathetic nervous system influences the musculoskeletal system by, for example, increasing osteoclastogenesis. This study was initiated to explore the role of the sympathetic neurotransmitter norepinephrine (NE) in mesenchymal stem cell (MSC)-dependent and cartilage progenitor cell (CPC)-dependent chondrogenesis. Using human MSCs or CPCs, chondrogenic differentiation was induced in the presence of NE, the specific β-adrenergic receptor (β-AR) agonist isoproterenol, and the specific β-AR antagonist nadolol. We studied sympathetic nerve fibers, tyrosine hydroxylase (TH) expression, catecholamine biosynthesis, and synovial fluid levels in human joints, as well as cartilage-specific matrix deposition during differentiation. TH+ sympathetic nerve fibers were present in the synovial tissue, meniscus, and subchondral bone marrow. In addition, synovial fluid from patients with knee trauma demonstrated high concentrations of NE. During MSC or CPC chondrogenesis, β-AR were expressed. Chondrogenic aggregates treated with NE or isoproterenol synthesized lower amounts of type II collagen and glycosaminoglycans. NE and isoproterenol treatment dose-dependently increased the levels of cartilage hypertrophy markers (type X collagen and matrix metalloproteinase 13). Nadolol reversed the inhibition of chondrogenesis and the up-regulation of cartilage hypertrophy. Our findings demonstrate NE-dependent inhibition of chondrogenesis and acceleration of hypertrophic differentiation. By inhibiting cartilage repair, these sympathetic influences can be important after joint trauma. These findings may be a basis for novel neurochondrogenic therapeutic options. Copyright © 2014 by the American College of

  3. Inhibition of Estradiol Synthesis Impairs Fear Extinction in Male Rats

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    Graham, Bronwyn M.; Milad, Mohammed R.

    2014-01-01

    Emerging research has demonstrated that the sex hormone estradiol regulates fear extinction in female rodents and women. Estradiol may also regulate fear extinction in males, given its role in synaptic plasticity in both sexes. Here we report that inhibition of estradiol synthesis during extinction training, via the aromatase inhibitor fadrozole,…

  4. Impairment of the chondrogenic phase of endochondral ossification in vivo by inhibition of cyclooxygenase-2

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    MPF Janssen

    2017-10-01

    Full Text Available Many studies have reported on the effects of cyclooxygenase-2 (COX-2 inhibition on osteogenesis. However, far less is known about the effects of COX-2 inhibition on chondrogenic differentiation. Previous studies conducted by our group show that COX-2 inhibition influences in vitro chondrogenic differentiation. Importantly, this might have consequences on endochondral ossification processes occurring in vivo, such as bone fracture healing, growth plate development and ectopic generation of cartilage. The goal of our study was to investigate, in vivo, the effect of COX-2 inhibition by celecoxib on the cartilaginous phase of three different endochondral ossification scenarios. 10 mg/kg/day celecoxib or placebo were orally administered for 25 d to skeletally-immature New Zealand White rabbits (n = 6 per group. Endochondral ossification during fracture healing of a non-critical size defect in the ulna, femoral growth plate and ectopically-induced cartilaginous tissue were examined by radiography, micro-computed tomography (µ-CT, histology and gene expression analysis. Celecoxib treatment resulted in delayed bone fracture healing, alterations in growth plate development and progression of mineralisation. In addition, chondrogenic differentiation of ectopically-induced cartilaginous tissue was severely impaired by celecoxib. In conclusion, we found that celecoxib impaired the chondrogenic phase of endochondral ossification.

  5. Interleukin-6 inhibits early differentiation of ATDC5 chondrogenic progenitor cells.

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    Nakajima, Shoko; Naruto, Takuya; Miyamae, Takako; Imagawa, Tomoyuki; Mori, Masaaki; Nishimaki, Shigeru; Yokota, Shumpei

    2009-08-01

    Interleukin (IL)-6 is a causative agent of systemic juvenile idiopathic arthritis (sJIA), a chronic inflammatory disease complicated with severe growth impairment. Recent trials of anti-IL-6 receptor monoclonal antibody, tocilizumab, indicated that tocilizumab blocks IL-6/IL-6 receptor-mediated inflammation, and induces catch-up growth in children with sJIA. This study evaluates the effects of IL-6 on chondrogenesis by ATDC5 cells, a clonal murine chondrogenic cell line that provides an excellent model for studying endochondral ossification at growth plate. ATDC5 cells were examined for the expression of IL-6 receptor and gp130 by fluorescence-activated cell sorting analysis. Recombinant murine IL-6 was added to ATDC5 cultures to observe cell differentiation, using a quantitative RT-PCR for the chondrogenic differentiation markers type II collagen, aggrecan, and type X collagen. To block IL-6, the anti-mouse IL-6 receptor monoclonal antibody MR16-1 was added. As a result, the cells expressed IL-6 receptor and gp130. The expression of chondrogenic differentiation marker gene was reduced by IL-6, but this was abrogated by MR16-1. We conclude that IL-6 inhibits early chondrogenesis of ATDC5 cells suggesting that IL-6 may affect committed stem cells at a cellular level during chondrogenic differentiation of growth plate chondrocytes, and that IL-6 may be a cellular-level factor in growth impairment in sJIA.

  6. Curcumin inhibits cellular condensation and alters microfilament organization during chondrogenic differentiation of limb bud mesenchymal cells

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    Kim, Dongkyun; Kim, Song-Ja; Kang, Shin-Sung; Jin, Eun-Jung

    2009-01-01

    Curcumin is a well known natural polyphenol product isolated from the rhizome of the plant Curcuma longa, anti-inflammatory agent for arthritis by inhibiting synthesis of inflammatory prostaglandins. However, the mechanisms by which curcumin regulates the functions of chondroprogenitor, such as proliferation, precartilage condensation, cytoskeletal organization or overall chondrogenic behavior, are largely unknown. In the present report, we investigated the effects and signaling mechanism of ...

  7. Curcumin inhibits cellular condensation and alters microfilament organization during chondrogenic differentiation of limb bud mesenchymal cells.

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    Kim, Dong Kyun; Kim, Song Ja; Kang, Shin Sung; Jin, Eun Jung

    2009-09-30

    Curcumin is a well known natural polyphenol product isolated from the rhizome of the plant Curcuma longa, anti-inflammatory agent for arthritis by inhibiting synthesis of inflammatory prostaglandins. However, the mechanisms by which curcumin regulates the functions of chondroprogenitor, such as proliferation, precartilage condensation, cytoskeletal organization or overall chondrogenic behavior, are largely unknown. In the present report, we investigated the effects and signaling mechanism of curcumin on the regulation of chondrogenesis. Treating chick limb bud mesenchymal cells with curcumin suppressed chondrogenesis by stimulating apoptotic cell death. It also inhibited reorganization of the actin cytoskeleton into a cortical pattern concomitant with rounding of chondrogenic competent cells and down-regulation of integrin beta1 and focal adhesion kinase (FAK) phosphorylation. Curcumin suppressed the phosphorylation of Akt leading to Akt inactivation. Activation of Akt by introducing a myristoylated, constitutively active form of Akt reversed the inhibitory actions of curcumin during chondrogenesis. In summary, for the first time, we describe biological properties of curcumin during chondrogenic differentiation of chick limb bud mesenchymal cells. Curcumin suppressed chondrogenesis by stimulating apoptotic cell death and down-regulating integrin-mediated reorganization of actin cytoskeleton via modulation of Akt signaling.

  8. Inhibition of TAK1 and/or JAK Can Rescue Impaired Chondrogenic Differentiation of Human Mesenchymal Stem Cells in Osteoarthritis-Like Conditions

    NARCIS (Netherlands)

    Beuningen, H.M. van; Vries-van Melle, M.L. de; Vitters, E.L.; Schreurs, W.; Berg, W.B. van den; Osch, G.J. van; Kraan, P.M. van der

    2014-01-01

    OBJECTIVE: To rescue chondrogenic differentiation of human mesenchymal stem cells (hMSCs) in osteoarthritic conditions by inhibition of protein kinases. METHODS: hMSCs were cultured in pellets. During early chondrogenic differentiation, these were exposed to osteoarthritic synovium-conditioned

  9. Extracellular Protease Inhibition Alters the Phenotype of Chondrogenically Differentiating Human Mesenchymal Stem Cells (MSCs) in 3D Collagen Microspheres.

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    Han, Sejin; Li, Yuk Yin; Chan, Barbara Pui

    2016-01-01

    Matrix remodeling of cells is highly regulated by proteases and their inhibitors. Nevertheless, how would the chondrogenesis of mesenchymal stem cells (MSCs) be affected, when the balance of the matrix remodeling is disturbed by inhibiting matrix proteases, is incompletely known. Using a previously developed collagen microencapsulation platform, we investigated whether exposing chondrogenically differentiating MSCs to intracellular and extracellular protease inhibitors will affect the extracellular matrix remodeling and hence the outcomes of chondrogenesis. Results showed that inhibition of matrix proteases particularly the extracellular ones favors the phenotype of fibrocartilage rather than hyaline cartilage in chondrogenically differentiating hMSCs by upregulating type I collagen protein deposition and type II collagen gene expression without significantly altering the hypertrophic markers at gene level. This study suggests the potential of manipulating extracellular proteases to alter the outcomes of hMSC chondrogenesis, contributing to future development of differentiation protocols for fibrocartilage tissues for intervertebral disc and meniscus tissue engineering.

  10. Extracellular Protease Inhibition Alters the Phenotype of Chondrogenically Differentiating Human Mesenchymal Stem Cells (MSCs in 3D Collagen Microspheres.

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    Sejin Han

    Full Text Available Matrix remodeling of cells is highly regulated by proteases and their inhibitors. Nevertheless, how would the chondrogenesis of mesenchymal stem cells (MSCs be affected, when the balance of the matrix remodeling is disturbed by inhibiting matrix proteases, is incompletely known. Using a previously developed collagen microencapsulation platform, we investigated whether exposing chondrogenically differentiating MSCs to intracellular and extracellular protease inhibitors will affect the extracellular matrix remodeling and hence the outcomes of chondrogenesis. Results showed that inhibition of matrix proteases particularly the extracellular ones favors the phenotype of fibrocartilage rather than hyaline cartilage in chondrogenically differentiating hMSCs by upregulating type I collagen protein deposition and type II collagen gene expression without significantly altering the hypertrophic markers at gene level. This study suggests the potential of manipulating extracellular proteases to alter the outcomes of hMSC chondrogenesis, contributing to future development of differentiation protocols for fibrocartilage tissues for intervertebral disc and meniscus tissue engineering.

  11. Estradiol receptors mediate estradiol-induced inhibition of mitochondrial Ca^{2+} efflux in rat caudate nucleus and brain stem

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    PETROVIC, SNJEZANA; MILOSEVIC, MAJA; RISTIC-MEDIC, DANIJELA; VELICKOVIC, NATASA; DRAKULIC, DUNJA; GRKOVIC, IVANA; HORVAT, ANICA

    2015-01-01

    Our earlier studies found that in vitro estradiol modulates mitochondrial Ca2+ transport in discrete brain regions. The present study examined the role of estradiol receptors (ERs) in estradiol-induced inhibition of Ca^{2+} efflux from synaptosomal mitochondria isolated from rat caudate nuclei and brain stems. Radioactively labeled CaCl_2 (0.6?0.75 µCi ^45CaCl_{2}) was used for Ca^{2+} transport monitoring. The results revealed that in the presence of ER antagonist 7\\alpha,17ß-[9[(4,4,5,5,5-...

  12. Delta-like 1/fetal antigen-1 (Dlk1/FA1) is a novel regulator of chondrogenic cell differentiation via inhibition of the Akt kinase-dependent pathway

    DEFF Research Database (Denmark)

    Chen, Li; Qanie, Diyako; Jafari, Abbas

    2011-01-01

    Delta-like 1 (Dlk1, also known as fetal antigen-1, FA1) is a member of Notch/Delta family that inhibits adipocyte and osteoblast differentiation; however, its role in chondrogenesis is still not clear. Thus, we overexpressed Dlk1/FA1 in mouse embryonic ATDC5 cells and tested its effects...... on chondrogenic differentiation. Dlk1/FA1 inhibited insulin-induced chondrogenic differentiation as evidenced by reduction of cartilage nodule formation and gene expression of aggrecan, collagen Type II and X. Similar effects were obtained either by using Dlk1/FA1-conditioned medium or by addition of a purified......, secreted, form of Dlk1 (FA1) directly to the induction medium. The inhibitory effects of Dlk1/FA1 were dose-dependent and occurred irrespective of the chondrogenic differentiation stage: proliferation, differentiation, maturation, or hypertrophic conversion. Overexpression or addition of the Dlk1/FA1...

  13. Delta-like 1/Fetal Antigen-1 (Dlk1/FA1) Is a Novel Regulator of Chondrogenic Cell Differentiation via Inhibition of the Akt Kinase-dependent Pathway*

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    Chen, Li; Qanie, Diyako; Jafari, Abbas; Taipaleenmaki, Hanna; Jensen, Charlotte H.; Säämänen, Anna-Marja; Sanz, Maria Luisa Nueda; Laborda, Jorge; Abdallah, Basem M.; Kassem, Moustapha

    2011-01-01

    Delta-like 1 (Dlk1, also known as fetal antigen-1, FA1) is a member of Notch/Delta family that inhibits adipocyte and osteoblast differentiation; however, its role in chondrogenesis is still not clear. Thus, we overexpressed Dlk1/FA1 in mouse embryonic ATDC5 cells and tested its effects on chondrogenic differentiation. Dlk1/FA1 inhibited insulin-induced chondrogenic differentiation as evidenced by reduction of cartilage nodule formation and gene expression of aggrecan, collagen Type II and X. Similar effects were obtained either by using Dlk1/FA1-conditioned medium or by addition of a purified, secreted, form of Dlk1 (FA1) directly to the induction medium. The inhibitory effects of Dlk1/FA1 were dose-dependent and occurred irrespective of the chondrogenic differentiation stage: proliferation, differentiation, maturation, or hypertrophic conversion. Overexpression or addition of the Dlk1/FA1 protein to the medium strongly inhibited the activation of Akt, but not the ERK1/2, or p38 MAPK pathways, and the inhibition of Akt by Dlk1/FA1 was mediated through PI3K activation. Interestingly, inhibition of fibronectin expression by siRNA rescued the Dlk1/FA1-mediated inhibition of Akt, suggesting interaction of Dlk1/FA1 and fibronectin in chondrogenic cells. Our results identify Dlk1/FA1 as a novel regulator of chondrogenesis and suggest Dlk1/FA1 acts as an inhibitor of the PI3K/Akt pathways that leads to its inhibitory effects on chondrogenesis. PMID:21724852

  14. Faslodex inhibits estradiol-induced extracellular matrix dynamics and lung metastasis in a model of lymphangioleiomyomatosis.

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    Li, Chenggang; Zhou, Xiaobo; Sun, Yang; Zhang, Erik; Mancini, John D; Parkhitko, Andrey; Morrison, Tasha A; Silverman, Edwin K; Henske, Elizabeth P; Yu, Jane J

    2013-07-01

    Lymphangioleiomyomatosis (LAM) is a destructive lung disease primarily affecting women. Genetic studies indicate that LAM cells carry inactivating tuberous sclerosis complex (TSC)-2 mutations, and metastasize to the lung. We previously discovered that estradiol increases the metastasis of TSC2-deficient cells in mice carrying xenograft tumors. Here, we investigate the molecular basis underlying the estradiol-induced lung metastasis of TSC2-deficient cells, and test the efficacy of Faslodex (an estrogen receptor antagonist) in a preclinical model of LAM. We used a xenograft tumor model in which estradiol induces the lung metastasis of TSC2-deficient cells. We analyzed the impact of Faslodex on tumor size, the extracellular matrix organization, the expression of matrix metalloproteinase (MMP)-2, and lung metastasis. We also examined the effects of estradiol and Faslodex on MMP2 expression and activity in tuberin-deficient cells in vitro. Estradiol resulted in a marked reduction of Type IV collagen deposition in xenograft tumors, associated with 2-fold greater MMP2 concentrations compared with placebo-treated mice. Faslodex normalized the Type IV collagen changes in xenograft tumors, enhanced the survival of the mice, and completely blocked lung metastases. In vitro, estradiol enhanced MMP2 transcripts, protein accumulation, and activity. These estradiol-induced changes in MMP2 were blocked by Faslodex. In TSC2-deficient cells, estradiol increased MMP2 concentrations in vitro and in vivo, and induced extracellular matrix remodeling. Faslodex inhibits the estradiol-induced lung metastasis of TSC2-deficient cells. Targeting estrogen receptors with Faslodex may be of efficacy in the treatment of LAM.

  15. Faslodex Inhibits Estradiol-Induced Extracellular Matrix Dynamics and Lung Metastasis in a Model of Lymphangioleiomyomatosis

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    Li, Chenggang; Zhou, Xiaobo; Sun, Yang; Zhang, Erik; Mancini, John D.; Parkhitko, Andrey; Morrison, Tasha A.; Silverman, Edwin K.; Henske, Elizabeth P.

    2013-01-01

    Lymphangioleiomyomatosis (LAM) is a destructive lung disease primarily affecting women. Genetic studies indicate that LAM cells carry inactivating tuberous sclerosis complex (TSC)–2 mutations, and metastasize to the lung. We previously discovered that estradiol increases the metastasis of TSC2-deficient cells in mice carrying xenograft tumors. Here, we investigate the molecular basis underlying the estradiol-induced lung metastasis of TSC2-deficient cells, and test the efficacy of Faslodex (an estrogen receptor antagonist) in a preclinical model of LAM. We used a xenograft tumor model in which estradiol induces the lung metastasis of TSC2-deficient cells. We analyzed the impact of Faslodex on tumor size, the extracellular matrix organization, the expression of matrix metalloproteinase (MMP)–2, and lung metastasis. We also examined the effects of estradiol and Faslodex on MMP2 expression and activity in tuberin-deficient cells in vitro. Estradiol resulted in a marked reduction of Type IV collagen deposition in xenograft tumors, associated with 2-fold greater MMP2 concentrations compared with placebo-treated mice. Faslodex normalized the Type IV collagen changes in xenograft tumors, enhanced the survival of the mice, and completely blocked lung metastases. In vitro, estradiol enhanced MMP2 transcripts, protein accumulation, and activity. These estradiol-induced changes in MMP2 were blocked by Faslodex. In TSC2-deficient cells, estradiol increased MMP2 concentrations in vitro and in vivo, and induced extracellular matrix remodeling. Faslodex inhibits the estradiol-induced lung metastasis of TSC2-deficient cells. Targeting estrogen receptors with Faslodex may be of efficacy in the treatment of LAM. PMID:23526212

  16. Transfection of the IHH gene into rabbit BMSCs in a simulated microgravity environment promotes chondrogenic differentiation and inhibits cartilage aging.

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    Liu, Peng-Cheng; Liu, Kuan; Liu, Jun-Feng; Xia, Kuo; Chen, Li-Yang; Wu, Xing

    2016-09-27

    The effect of overexpressing the Indian hedgehog (IHH) gene on the chondrogenic differentiation of rabbit bone marrow-derived mesenchymal stem cells (BMSCs) was investigated in a simulated microgravity environment. An adenovirus plasmid encoding the rabbit IHH gene was constructed in vitro and transfected into rabbit BMSCs. Two large groups were used: conventional cell culture and induction model group and simulated microgravity environment group. Each large group was further divided into blank control group, GFP transfection group, and IHH transfection group. During differentiation induction, the expression levels of cartilage-related and cartilage hypertrophy-related genes and proteins in each group were determined. In the conventional model, the IHH transfection group expressed high levels of cartilage-related factors (Coll2 and ANCN) at the early stage of differentiation induction and expressed high levels of cartilage hypertrophy-related factors (Coll10, annexin 5, and ALP) at the late stage. Under the simulated microgravity environment, the IHH transfection group expressed high levels of cartilage-related factors and low levels of cartilage hypertrophy-related factors at all stages of differentiation induction. Under the simulated microgravity environment, transfection of the IHH gene into BMSCs effectively promoted the generation of cartilage and inhibited cartilage aging and osteogenesis. Therefore, this technique is suitable for cartilage tissue engineering.

  17. Estradiol inhibits hepatic stellate cell area and collagen synthesis in the chicken liver.

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    Nishimura, Shotaro; Teshima, Akifumi; Kawabata, Fuminori; Tabata, Shoji

    2017-11-01

    Hepatic stellate cells (HSCs) are the main collagen-producing cells in the liver. The HSC area and amount of collagen fibers are different between male and female chickens. This study was performed to confirm the effect of estradiol on collagen synthesis in the growing chicken liver. Blood estradiol levels in chicks were compared at 4 and 8 weeks of age, and the collagen fibril network in liver tissue was observed at 8 weeks by scanning electron microscopy. Intraperitoneal administrations of estradiol and tamoxifen to male and female chicks, respectively, were performed daily from 5 to 8 weeks of age. The areas of HSCs and collagen contents were measured in the liver tissue. The blood estradiol level was higher in females than in males, and the collagen fibril network was denser in males than in females at 8 weeks of age. Estradiol administration in males induced decreases in the HSC area and collagen content of the liver. Conversely, tamoxifen administration in females induced an increase in the HSC area but did not facilitate collagen synthesis. Based on these results, estradiol inhibits the area and collagen synthesis of HSCs in the growing chicken liver under normal physiological conditions. © 2017 Japanese Society of Animal Science.

  18. Estradiol-induced vaginal mucus inhibits antigen penetration and CD8(+) T cell priming in response to intravaginal immunization.

    Science.gov (United States)

    Seavey, Matthew M; Mosmann, Tim R

    2009-04-14

    Although vaginal immunization has been explored as a strategy to induce mucosal immunity in the female reproductive tract, this site displays unique immunological features that probably evolved to inhibit anti-paternal T cell responses after insemination to allow successful pregnancy. We previously demonstrated that estradiol, which induces an estrus-like state, prevented CD8(+) T cell priming during intravaginal immunization of mice. We now show that estradiol prevented antigen loading of vaginal antigen presenting cells (APCs) after intravaginal immunization. Histological examination confirmed that estradiol prevented penetration of peptide antigen into the vaginal wall. Removal of the estradiol-induced mucus barrier by mucinase partially restored antigen loading of vaginal APC and CD8(+) T cell proliferation in vivo. The estradiol-induced mucus barrier may thus prevent exposure to antigens delivered intravaginally, supplementing additional estradiol-dependent mechanism(s) that inhibit CD8(+) T cell priming after insemination or vaginal vaccination.

  19. Estradiol-induced vaginal mucus inhibits antigen penetration and CD8+ T cell priming in response to intravaginal immunization

    Science.gov (United States)

    Seavey, Matthew M.; Mosmann, Tim R.

    2010-01-01

    Although vaginal immunization has been explored as a strategy to induce mucosal immunity in the female reproductive tract, this site displays unique immunological features that probably evolved to inhibit anti-paternal T cell responses after insemination to allow successful pregnancy. We previously demonstrated that estradiol, which induces an estrus-like state, prevented CD8+ T cell priming during intravaginal immunization of mice. We now show that estradiol prevented antigen loading of vaginal antigen presenting cells (APC) after intravaginal immunization. Histological examination confirmed that estradiol prevented penetration of peptide antigen into the vaginal wall. Removal of the estradiol-induced mucus barrier by mucinase partially restored antigen loading of vaginal APC and CD8+ T cell proliferation in vivo. The estradiol-induced mucus barrier may thus prevent exposure to antigens delivered intravaginally, supplementing additional estradiol-dependent mechanism(s) that inhibit CD8+ T cell priming after insemination or vaginal vaccination. PMID:19428849

  20. Estradiol and Progesterone Strongly Inhibit the Innate Immune Response of Mononuclear Cells in Newborns ▿

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    Giannoni, Eric; Guignard, Laurence; Knaup Reymond, Marlies; Perreau, Matthieu; Roth-Kleiner, Matthias; Calandra, Thierry; Roger, Thierry

    2011-01-01

    Newborns are particularly susceptible to bacterial infections due to qualitative and quantitative deficiencies of the neonatal innate immune system. However, the mechanisms underlying these deficiencies are poorly understood. Given that fetuses are exposed to high concentrations of estradiol and progesterone during gestation and at time of delivery, we analyzed the effects of these hormones on the response of neonatal innate immune cells to endotoxin, bacterial lipopeptide, and Escherichia coli and group B Streptococcus, the two most common causes of early-onset neonatal sepsis. Here we show that at concentrations present in umbilical cord blood, estradiol and progesterone are as powerful as hydrocortisone for inhibition of cytokine production by cord blood mononuclear cells (CBMCs) and newborn monocytes. Interestingly, CBMCs and newborn monocytes are more sensitive to the effects of estradiol and progesterone than adult peripheral blood mononuclear cells and monocytes. This increased sensitivity is associated with higher expression levels of estrogen and membrane progesterone receptors but is independent of a downregulation of Toll-like receptor 2 (TLR2), TLR4, and myeloid differentiation primary response gene 88 in newborn cells. Estradiol and progesterone mediate their anti-inflammatory activity through inhibition of the NF-κB pathway but not the mitogen-activated protein kinase pathway in CBMCs. Altogether, these results suggest that elevated umbilical cord blood concentrations of estradiol and progesterone acting on mononuclear cells expressing high levels of steroid receptors contribute to impair innate immune responses in newborns. Therefore, intrauterine exposure to estradiol and progesterone may participate in increasing susceptibility to infection during the neonatal period. PMID:21518785

  1. MiR-125b Inhibits LPS-Induced Inflammatory Injury via Targeting MIP-1α in Chondrogenic Cell ATDC5

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    Jinling Jia

    2018-03-01

    Full Text Available Background/Aims: Chondrocyte apoptosis is largely responsible for cartilage degeneration in osteoarthritis (OA. MicroRNAs (miRNAs play an important role in chondrogenesis and cartilage remodeling. This study explored the effect of miR-125b on inflammatory injury in chondrogenic cells. Methods: LPS was used to simulate inflammatory injury in murine chondrogenic ATDC5 cell lines. Targeting effect of miR-125b on MIP-1α 3’UTR was assessed by dual luciferase activity assay. Regulatory effect of miR-125b on MIP-1α expression and the potential regulatory mechanism on inflammatory injury were assessed by Western blot. Results: miR-125b expression was decreased in LPS-induced ATDC5 cells and overexpression of miR-125b inhibited LPS-induced cell viability decline, the rise of apoptosis and inflammatory factors’ productions. MIP-1α expression was negatively related to miR-125b, and miR-125b directly targeted with 3’UTR of MIP-1α. Knockdown of miR-125b promoted LPS-induced inflammatory response via upregulation of MIP-1α. miR-125b expression in LPS-induced ATDC5 cells was negatively related with activations of NF-κB and JNK signaling pathways. Overexpression of miR-125b inhibited LPS-induced inflammation injury via suppressing MIP-1α expression and inhibiting activations of NF-κB and JNK signaling pathways. Conclusion: miR-125b could play an important role in inflammatory injury of chondrogenic cells and miR-125b affected inflammatory injury of ATDC5 cells via regulating expression of MIP-1α and regulating NF-κB and JNK signaling pathways.

  2. Diethylstilbestrol can effectively accelerate estradiol-17-O-glucuronidation, while potently inhibiting estradiol-3-O-glucuronidation

    International Nuclear Information System (INIS)

    Zhu, Liangliang; Xiao, Ling; Xia, Yangliu; Zhou, Kun; Wang, Huili; Huang, Minyi; Ge, Guangbo; Wu, Yan; Wu, Ganlin; Yang, Ling

    2015-01-01

    This in vitro study investigates the effects of diethylstilbestrol (DES), a widely used toxic synthetic estrogen, on estradiol-3- and 17-O- (E2-3/17-O) glucuronidation, via culturing human liver microsomes (HLMs) or recombinant UDP-glucuronosyltransferases (UGTs) with DES and E2. DES can potently inhibit E2-3-O-glucuronidation in HLM, a probe reaction for UGT1A1. Kinetic assays indicate that the inhibition follows a competitive inhibition mechanism, with the Ki value of 2.1 ± 0.3 μM, which is less than the possible in vivo level. In contrast to the inhibition on E2-3-O-glucuronidation, the acceleration is observed on E2-17-O-glucuronidation in HLM, in which cholestatic E2-17-O-glucuronide is generated. In the presence of DES (0–6.25 μM), K m values for E2-17-O-glucuronidation are located in the range of 7.2–7.4 μM, while V max values range from 0.38 to 1.54 nmol/min/mg. The mechanism behind the activation in HLM is further demonstrated by the fact that DES can efficiently elevate the activity of UGT1A4 in catalyzing E2-17-O-glucuronidation. The presence of DES (2 μM) can elevate V max from 0.016 to 0.81 nmol/min/mg, while lifting K m in a much lesser extent from 4.4 to 11 μM. Activation of E2-17-O-glucuronidation is well described by a two binding site model, with K A , α, and β values of 0.077 ± 0.18 μM, 3.3 ± 1.1 and 104 ± 56, respectively. However, diverse effects of DES towards E2-3/17-O-glucuronidation are not observed in liver microsomes from several common experimental animals. In summary, this study issues new potential toxic mechanisms for DES: potently inhibiting the activity of UGT1A1 and powerfully accelerating the formation of cholestatic E2-17-O-glucuronide by UGT1A4. - Highlights: • E2-3-O-glucuronidation in HLM is inhibited when co-incubated with DES. • E2-17-O-glucuronidation in HLM is stimulated when co-incubated with DES. • Acceleration of E2-17-O-glucuronidationin in HLM by DES is via activating the activity of UGT1A4

  3. Histone deacetylase 4 promotes TGF-beta1-induced synovium-derived stem cell chondrogenesis but inhibits chondrogenically differentiated stem cell hypertrophy.

    Science.gov (United States)

    Pei, Ming; Chen, Demeng; Li, Jingting; Wei, Lei

    2009-12-01

    The transforming growth factor-beta (TGF-beta) superfamily members play diverse roles in cartilage development and maintenance. TGF-beta up-regulates chondrogenic gene expression by enhancing transcription factor SRY (sex determining region Y)-box 9 (Sox9) and inhibits osteoblast differentiation by repressing runt-related transcription factor 2 (Runx2). Recently, histone deacetylases (HDACs) were reported to act as negative regulators of chondrocyte hypertrophy. It was speculated that HDAC4 may promote TGF-beta1-induced MSC chondrogenesis. In this study, the adenovirus-mediated HDAC4 gene (Ad.HDAC4) was utilized to infect synovium-derived stem cells (SDSCs). Adenovirus-mediated LacZ (Ad.LacZ) served as a control. The infected cells were centrifuged to form SDSC pellets followed by incubation in a serum-free chondrogenic medium for 15 days with or without 10ng/mL TGF-beta1. Transfection efficiency was determined in SDSCs using Ad.LacZ. Cytotoxicity was measured using lactate dehydrogenase assay. Histology, immunostaining, biochemical analysis, and real-time polymerase chain reaction were performed to assess chondrogenesis at protein and mRNA levels in infected SDSCs. Our data demonstrated that supplementation with TGF-beta1 could initiate and promote SDSC chondrogenesis; however, TGF-beta1 alone was insufficient to fully differentiate SDSCs into chondrocytes. Ad.HDAC4 could be efficiently transfected into SDSCs. Without TGF-beta1 treatment, HDAC4 had no effect on SDSC chondrogenesis; however, in the presence of TGF-beta1, HDAC4 could speed up and maintain a high level of chondrogenesis while down-regulating the hypertrophic marker - type X collagen expression. This study is the first report showing that HDAC4 overexpression promotes TGF-beta1-induced SDSC chondrogenesis but inhibits chondrogenically differentiated stem cell hypertrophy. The mechanism underlying this process needs further investigation.

  4. Di-(2-ethylhexyl) phthalate and mono-(2-ethylhexyl) phthalate inhibit growth and reduce estradiol levels of antral follicles in vitro

    International Nuclear Information System (INIS)

    Gupta, Rupesh K.; Singh, Jeffery M.; Leslie, Tracie C.; Meachum, Sharon; Flaws, Jodi A.; Yao, Humphrey H-C

    2010-01-01

    Any insult that affects survival of ovarian antral follicles can cause abnormal estradiol production and fertility problems. Phthalate esters (PEs) are plasticizers used in a wide range of consumer and industrial products. Exposure to these chemicals has been linked to reduced fertility in humans and animal models. Di-(2-ethylhexyl) phthalate (DEHP) and mono-(2-ethylhexyl) phthalate (MEHP) decrease serum estradiol levels and aromatase (Arom) expression, prolong estrous cycles, and cause anovulation in animal and culture models. These observations suggest PEs directly target antral follicles. We therefore tested the hypothesis that DEHP (1-100 μg/ml) and MEHP (0.1-10 μg/ml) directly inhibit antral follicular growth and estradiol production. Antral follicles from adult mice were cultured with DEHP or MEHP, and/or estradiol for 96 h. During culture, follicle size was measured every 24 h as a measurement of follicle growth. After culture, media were collected for measurement of estradiol levels and follicles were subjected to measurement of cylin-D-2 (Ccnd2), cyclin-dependant-kinase-4 (Cdk4), and Arom. We found that DEHP and MEHP inhibited growth of follicles and decreased estradiol production compared to controls at the highest doses. DEHP and MEHP also decreased mRNA expression of Ccnd2, Cdk4, and Arom at the highest dose. Addition of estradiol to the culture medium prevented the follicles from DEHP- and MEHP-induced inhibition of growth, reduction in estradiol levels, and decreased Ccnd2 and Cdk4 expression. Collectively, our results indicate that DEHP and MEHP may directly inhibit antral follicle growth via a mechanism that partially includes reduction in levels of estradiol production and decreased expression of cell cycle regulators.

  5. Diethylstilbestrol can effectively accelerate estradiol-17-O-glucuronidation, while potently inhibiting estradiol-3-O-glucuronidation

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Liangliang; Xiao, Ling [The Centre for Drug and Food Safety Evaluation, School of Life Science, Anqing Normal University, Anqing 246011 (China); Xia, Yangliu [Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023 (China); Zhou, Kun [College of Pharmacy, Liaoning University of Traditional Chinese Medicine, Dalian 116600 (China); Wang, Huili; Huang, Minyi [The Centre for Drug and Food Safety Evaluation, School of Life Science, Anqing Normal University, Anqing 246011 (China); Ge, Guangbo, E-mail: geguangbo@dicp.ac.cn [Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023 (China); Wu, Yan; Wu, Ganlin [The Centre for Drug and Food Safety Evaluation, School of Life Science, Anqing Normal University, Anqing 246011 (China); Yang, Ling, E-mail: yling@dicp.ac.cn [Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023 (China)

    2015-03-01

    This in vitro study investigates the effects of diethylstilbestrol (DES), a widely used toxic synthetic estrogen, on estradiol-3- and 17-O- (E2-3/17-O) glucuronidation, via culturing human liver microsomes (HLMs) or recombinant UDP-glucuronosyltransferases (UGTs) with DES and E2. DES can potently inhibit E2-3-O-glucuronidation in HLM, a probe reaction for UGT1A1. Kinetic assays indicate that the inhibition follows a competitive inhibition mechanism, with the Ki value of 2.1 ± 0.3 μM, which is less than the possible in vivo level. In contrast to the inhibition on E2-3-O-glucuronidation, the acceleration is observed on E2-17-O-glucuronidation in HLM, in which cholestatic E2-17-O-glucuronide is generated. In the presence of DES (0–6.25 μM), K{sub m} values for E2-17-O-glucuronidation are located in the range of 7.2–7.4 μM, while V{sub max} values range from 0.38 to 1.54 nmol/min/mg. The mechanism behind the activation in HLM is further demonstrated by the fact that DES can efficiently elevate the activity of UGT1A4 in catalyzing E2-17-O-glucuronidation. The presence of DES (2 μM) can elevate V{sub max} from 0.016 to 0.81 nmol/min/mg, while lifting K{sub m} in a much lesser extent from 4.4 to 11 μM. Activation of E2-17-O-glucuronidation is well described by a two binding site model, with K{sub A}, α, and β values of 0.077 ± 0.18 μM, 3.3 ± 1.1 and 104 ± 56, respectively. However, diverse effects of DES towards E2-3/17-O-glucuronidation are not observed in liver microsomes from several common experimental animals. In summary, this study issues new potential toxic mechanisms for DES: potently inhibiting the activity of UGT1A1 and powerfully accelerating the formation of cholestatic E2-17-O-glucuronide by UGT1A4. - Highlights: • E2-3-O-glucuronidation in HLM is inhibited when co-incubated with DES. • E2-17-O-glucuronidation in HLM is stimulated when co-incubated with DES. • Acceleration of E2-17-O-glucuronidationin in HLM by DES is via activating the

  6. Inhibition of the mitochondrial enzyme ABAD restores the amyloid-β-mediated deregulation of estradiol.

    Directory of Open Access Journals (Sweden)

    Yun-An Lim

    Full Text Available Alzheimer's disease (AD is a conformational disease that is characterized by amyloid-β (Aβ deposition in the brain. Aβ exerts its toxicity in part by receptor-mediated interactions that cause down-stream protein misfolding and aggregation, as well as mitochondrial dysfunction. Recent reports indicate that Aβ may also interact directly with intracellular proteins such as the mitochondrial enzyme ABAD (Aβ binding alcohol dehydrogenase in executing its toxic effects. Mitochondrial dysfunction occurs early in AD, and Aβ's toxicity is in part mediated by inhibition of ABAD as shown previously with an ABAD decoy peptide. Here, we employed AG18051, a novel small ABAD-specific compound inhibitor, to investigate the role of ABAD in Aβ toxicity. Using SH-SY5Y neuroblastoma cells, we found that AG18051 partially blocked the Aβ-ABAD interaction in a pull-down assay while it also prevented the Aβ42-induced down-regulation of ABAD activity, as measured by levels of estradiol, a known hormone and product of ABAD activity. Furthermore, AG18051 is protective against Aβ42 toxicity, as measured by LDH release and MTT absorbance. Specifically, AG18051 reduced Aβ42-induced impairment of mitochondrial respiration and oxidative stress as shown by reduced ROS (reactive oxygen species levels. Guided by our previous finding of shared aspects of the toxicity of Aβ and human amylin (HA, with the latter forming aggregates in Type 2 diabetes mellitus (T2DM pancreas, we determined whether AG18051 would also confer protection from HA toxicity. We found that the inhibitor conferred only partial protection from HA toxicity indicating distinct pathomechanisms of the two amyloidogenic agents. Taken together, our results present the inhibition of ABAD by compounds such as AG18051 as a promising therapeutic strategy for the prevention and treatment of AD, and suggest levels of estradiol as a suitable read-out.

  7. [27- Hydroxycholesterol reverses estradiol induced inhibition of platelet aggregation in postmenopausal women].

    Science.gov (United States)

    Rocha, Gladys; Sierralta, Walter; Valladares, Luis

    2016-11-01

    The decline of estrogen levels increases cardiovascular risk in women. Platelets express estrogen receptors and 17β-estradiol- (E2) can produce a protective effect on thrombus formation. The hydroxylation of cholesterol generates several sterols and 27-hydroxycholesterol (27HC) predominates in circulation. To evaluate the effect of 27HC as an endogenous antagonist of the anti-aggregating properties of E2 in platelets of postmenopausal women. Platelet function of postmenopausal women was evaluated ex-vivo. Platelets pre-incubated with 27HC in the presence or absence of E2, were stimulated with collagen. Aggregation was evaluated using turbidimetry using a Chrono-log aggregometer. Collagen-stimulated platelet aggregation was significantly inhibited by E2. The inhibitory effect of E2 on collagen-stimulated platelet aggregation was significantly reversed in the presence of 27HC. The suppressive effect of E2 on platelet aggregation is inhibited by 27HC, which could contribute to increase cardiovascular risk in postmenopausal women.

  8. Sox9-regulated miRNA-574-3p inhibits chondrogenic differentiation of mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    David Guérit

    Full Text Available The aim of this study was to identify new microRNAs (miRNAs that are modulated during the differentiation of mesenchymal stem cells (MSCs toward chondrocytes. Using large scale miRNA arrays, we compared the expression of miRNAs in MSCs (day 0 and at early time points (day 0.5 and 3 after chondrogenesis induction. Transfection of premiRNA or antagomiRNA was performed on MSCs before chondrogenesis induction and expression of miRNAs and chondrocyte markers was evaluated at different time points during differentiation by RT-qPCR. Among miRNAs that were modulated during chondrogenesis, we identified miR-574-3p as an early up-regulated miRNA. We found that miR-574-3p up-regulation is mediated via direct binding of Sox9 to its promoter region and demonstrated by reporter assay that retinoid X receptor (RXRα is one gene specifically targeted by the miRNA. In vitro transfection of MSCs with premiR-574-3p resulted in the inhibition of chondrogenesis demonstrating its role during the commitment of MSCs towards chondrocytes. In vivo, however, both up- and down-regulation of miR-574-3p expression inhibited differentiation toward cartilage and bone in a model of heterotopic ossification. In conclusion, we demonstrated that Sox9-dependent up-regulation of miR-574-3p results in RXRα down-regulation. Manipulating miR-574-3p levels both in vitro and in vivo inhibited chondrogenesis suggesting that miR-574-3p might be required for chondrocyte lineage maintenance but also that of MSC multipotency.

  9. Deficits in latent inhibition induced by estradiol replacement are ameliorated by haloperidol treatment

    Directory of Open Access Journals (Sweden)

    Anne eAlmey

    2013-10-01

    Full Text Available There are sex differences in the symptomatology of schizophrenia, and in the response to antipsychotic treatments. One hallmark symptom of schizophrenia is a deficit in selective attention. Selective attention can be measured using a latent inhibition (LI paradigm in humans; LI can be measured in rodents, and is used as an animal model of the selective attention deficits observed in schizophrenia. In the current experiments LI was used to clarify whether selective attention differs between male rats and ovariectomized (OVX female rats receiving different estradiol (E2 replacement regimens. An additional aim was to determine whether haloperidol's facilitation of LI is enhanced by E2. Males and OVX female rats were trained in a conditioned emotional response LI paradigm. Females received no E2 replacement, a chronic low dose of E2 via silastic capsule, or a high phasic dose of E2 via silastic capsule accompanied by E2 (10 ug/kg SC injections every fourth day. Actual plasma levels of E2 were determined using an enzyme linked immunosorbent assay. Rats were also administered a vehicle treatment, a 0.05mg/kg, or a 0.1mg/kg IP injection of haloperidol. Males and OVX females that did not receive E2 replacement both exhibited LI, but LI was not observed in the low and high E2 replacement groups. Haloperidol restored LI at a lower dose in the females receiving high E2 replacement compared to females receiving low E2 replacement, indicating that E2 replacement facilitates haloperidol in restoring LI.

  10. A peptide derived from alpha-fetoprotein inhibits the proliferation induced by estradiol in mammary tumor cells in culture.

    Science.gov (United States)

    Sierralta, Walter D; Epuñan, Maria J; Reyes, José M; Valladares, Luis E; Andersen, Thomas T; Bennett, James A; Jacobson, Herbert I; Pino, Ana M

    2008-01-01

    This study was aimed to obtain additional information on the activity of a cyclized 9-amino acid peptide (cP) containing the active site of alpha fetoprotein, which inhibits the estrogen-stimulated proliferation of tumor cells in culture and of xenografts in immunodeficient mice. Breast cancer cells cultured in the presence of 2 nM estradiol were exposed to cP for different periods and their proliferation, estradiol binding parameters, clustering tendency and expression of E-cadherin and p21Cip1 were analyzed by biochemical and cell biology methods. The proliferation of MCF7 cells was significantly decreased by the addition of 2 microg/ml cP to the medium. cP did not increase cell death rate nor alter the number of binding sites for estradiol nor the endogenous aromatase activity of MCF7 cells. cP also decreased the proliferation of estrogen-dependent ZR75-1 cells but had no effect on estrogen-independent MDA-MB-231 cells. An increased nuclear p21Cip1 expression detected after cP treatment suggests that cP slows MCF7 cell proliferation via this regulator. We propose that cP could represent a novel breast cancer therapeutic agent whose mechanism of action is different from that of tamoxifen or of inhibitors of aromatase.

  11. Estradiol agonists inhibit human LoVo colorectal-cancer cell proliferation and migration through p53.

    Science.gov (United States)

    Hsu, Hsi-Hsien; Kuo, Wei-Wen; Ju, Da-Tong; Yeh, Yu-Lan; Tu, Chuan-Chou; Tsai, Ying-Lan; Shen, Chia-Yao; Chang, Sheng-Huang; Chung, Li-Chin; Huang, Chih-Yang

    2014-11-28

    To investigate the effects of 17β-estradiol via estrogen receptors (ER) or direct administration of ER agonists on human colorectal cancer. LoVo cells were established from the Bioresource Collection and Research Center and cultured in phenol red-free DMEM (Sigma, United States). To investigate the effects of E2 and/or ER selective agonists on cellular proliferation, LoVo colorectal cells were treated with E2 or ER-selective agonists for 24 h and 48 h and subjected to the MTT (Sigma) assay to find the concentration. And investigate the effects of E2 and/or ER selective agonists on cell used western immunoblotting to find out the diversification of signaling pathways. In order to observe motility and migration the wound healing assay and a transwell chamber (Neuro Probe) plate were tased. For a quantitative measure, we counted the number of migrating cells to the wound area post-wounding for 24 h. We further examined the cellular migration-regulating factors urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA) and matrix metalloproteinase (MMP)-9 in human LoVo cells so gelatin zymography that we used and gelatinolytic activity was visualized by Coomassie blue staining. And these results are presented as means ± SE, and statistical comparisons were made using Student's t-test. The structure was first compared with E2 and ER agonists. We then treated the LoVo cells with E2 and ER agonists (10(-8) mol/L) for 24 h and 48 h and subsequently measured the cell viability using MTT assay. Our results showed that treatment with 17β-estradiol and/or ER agonists in human LoVo colorectal cancer cells activated p53 and then up-regulated p21 and p27 protein levels, subsequently inhibiting the downstream target gene, cyclin D1, which regulates cell proliferation. Taken together, our findings demonstrate the anti-tumorigenesis effects of 17β-estradiol and/or ER agonists and suggest that these compounds may prove to be a potential alternative

  12. Effects of 17β-estradiol on radiation transformation in vitro; inhibition of effects by protease inhibitors

    International Nuclear Information System (INIS)

    Kennedy, A.R.; Weichselbaum, R.R.

    1981-01-01

    The effects of 17β-estradiol, given either alone or with X-radiation, on the induction of malignant transformation were investigated in vitro. Treatment with 10 -6 M 17β-estradiol for 6 weeks, or 10 -5 M 17β-estradiol for only 5 days, induced malignant transformation in C3H 10T1/2 cells. Estradiol also acted as a cocarcinogen for X-ray induced transformation; the results indicated an additive effect when the cells were exposed to both agents together. The protease inhibitors antipain and leupeptin suppressed estradiol induced transformation as well as the additive effect observed for estradiol-radiation transformation. (author)

  13. Resveratrol protects the ovary against chromium-toxicity by enhancing endogenous antioxidant enzymes and inhibiting metabolic clearance of estradiol

    International Nuclear Information System (INIS)

    Banu, Sakhila K.; Stanley, Jone A.; Sivakumar, Kirthiram K.; Arosh, Joe A.; Burghardt, Robert C.

    2016-01-01

    Resveratrol (RVT), a polyphenolic component in grapes and red wine, has been known for its cytoprotective actions against several diseases. However, beneficial effects of RVT against early exposure to endocrine disrupting chemicals (EDCs) have not been understood. EDCs are linked to several ovarian diseases such as premature ovarian failure, polycystic ovary syndrome, early menopause and infertility in women. Hexavalent chromium (CrVI) is a heavy metal EDC, and widely used in > 50 industries. Environmental contamination with CrVI in the US is rapidly increasing, predisposing the human to several illnesses including cancers and still birth. Our lab has been involved in determining the molecular mechanism of CrVI-induced female infertility and intervention strategies to mitigate CrVI effects. Lactating mother rats were exposed to CrVI (50 ppm potassium dichromate) from postpartum days 1–21 through drinking water with or without RVT (10 mg/kg body wt., through oral gavage daily). During this time, F1 females received respective treatments through mother's milk. On postnatal day (PND) 25, blood and the ovary, kidney and liver were collected from the F1 females for analyses. CrVI increased atresia of follicles by increasing cytochrome C and cleaved caspase-3; decreasing antiapoptotic proteins; decreasing estradiol (E 2 ) biosynthesis and enhancing metabolic clearance of E 2 , increasing oxidative stress and decreasing endogenous antioxidants. RVT mitigated the effects of CrVI by upregulating cell survival proteins and AOXs; and restored E 2 levels by inhibiting hydroxylation, glucuronidation and sulphation of E 2 . This is the first study to report the protective effects of RVT against any toxicant in the ovary. - Highlights: • Resveratrol (RVT) protects the ovary against CrVI-toxicity. • RVT mitigated CrVI-induced apoptosis and follicle atresia. • RVT restored estradiol level against CrVI-toxicity. • RVT inhibited metabolic clearance of estradiol in the

  14. Resveratrol protects the ovary against chromium-toxicity by enhancing endogenous antioxidant enzymes and inhibiting metabolic clearance of estradiol

    Energy Technology Data Exchange (ETDEWEB)

    Banu, Sakhila K., E-mail: skbanu@cvm.tamu.edu; Stanley, Jone A.; Sivakumar, Kirthiram K.; Arosh, Joe A.; Burghardt, Robert C.

    2016-07-15

    Resveratrol (RVT), a polyphenolic component in grapes and red wine, has been known for its cytoprotective actions against several diseases. However, beneficial effects of RVT against early exposure to endocrine disrupting chemicals (EDCs) have not been understood. EDCs are linked to several ovarian diseases such as premature ovarian failure, polycystic ovary syndrome, early menopause and infertility in women. Hexavalent chromium (CrVI) is a heavy metal EDC, and widely used in > 50 industries. Environmental contamination with CrVI in the US is rapidly increasing, predisposing the human to several illnesses including cancers and still birth. Our lab has been involved in determining the molecular mechanism of CrVI-induced female infertility and intervention strategies to mitigate CrVI effects. Lactating mother rats were exposed to CrVI (50 ppm potassium dichromate) from postpartum days 1–21 through drinking water with or without RVT (10 mg/kg body wt., through oral gavage daily). During this time, F1 females received respective treatments through mother's milk. On postnatal day (PND) 25, blood and the ovary, kidney and liver were collected from the F1 females for analyses. CrVI increased atresia of follicles by increasing cytochrome C and cleaved caspase-3; decreasing antiapoptotic proteins; decreasing estradiol (E{sub 2}) biosynthesis and enhancing metabolic clearance of E{sub 2}, increasing oxidative stress and decreasing endogenous antioxidants. RVT mitigated the effects of CrVI by upregulating cell survival proteins and AOXs; and restored E{sub 2} levels by inhibiting hydroxylation, glucuronidation and sulphation of E{sub 2}. This is the first study to report the protective effects of RVT against any toxicant in the ovary. - Highlights: • Resveratrol (RVT) protects the ovary against CrVI-toxicity. • RVT mitigated CrVI-induced apoptosis and follicle atresia. • RVT restored estradiol level against CrVI-toxicity. • RVT inhibited metabolic clearance of

  15. Pueraria mirifica inhibits 17β-estradiol-induced cell proliferation of human endometrial mesenchymal stem cells.

    Science.gov (United States)

    Lin, Ta-Chin; Wang, Kai-Hung; Kao, An-Pei; Chuang, Kuo-Hsiang; Kuo, Tsung-Cheng

    2017-12-01

    The notion that the human endometrium may contain a population of stem cells has recently been proposed. The mesenchymal stem cells (MSCs) in the endometrium are believed to be responsible for the remarkable regenerative ability of endometrial cells. Estrogens influence the physiological and pathological processes of several hormone-dependent tissues, such as the endometrium. Pueraria mirifica (PM) is a herbal plant that contains several phytoestrogens, including isoflavones, lignans, and coumestans, and is known to exert an estrogenic effect on animal models. The present study investigated the effects of PM on the proliferation of human endometrial MSCs (hEN-MSCs). The hEN-MSCs were isolated from human endometrial tissue. The surface markers of these hEN-MSCs were identified through reverse transcription-polymerase chain reaction analysis. The proliferation potential of hEN-MSCs was measured through a cell proliferation assay. Multilineage differentiation ability was confirmed through Oil red O and von Kossa staining. This study demonstrated that 17β-estradiol-responsive MSCs with Oct-4, CD90, and CD105 gene expression can be derived from the human endometrium and that PM exerts biological effects on hEN-MSCs, specifically, enhanced cell growth rate, through the estrogen receptor. Furthermore, PM at 1500 and 2000 μg/mL significantly increased cell proliferation compared with the vehicle control, and PM concentration at 1000 μg/mL significantly inhibited the enhanced cell growth rate induced by 17β-estradiol in hEN-MSCs. This study provides new insights into the possible biological effects of PM on the proliferation of hEN-MSCs. Copyright © 2017. Published by Elsevier B.V.

  16. Black cohosh inhibits 17β-estradiol-induced cell proliferation of endometrial adenocarcinoma cells.

    Science.gov (United States)

    Park, So Yun; Kim, Hee Ja; Lee, Sa Ra; Choi, Youn-Hee; Jeong, Kyungah; Chung, Hyewon

    2016-10-01

    This study was conducted to investigate the effect of black cohosh (BC) extract on the proliferation and apoptosis of Ishikawa cells. Ishikawa human endometrial adenocarcinoma cells were treated with or without BC (1, 5, 10 and 25 μM) and cell proliferation and cytotoxicity were measured by CCK-8 assays and flow cytometry analysis. Additionally, Ishikawa cells were treated with 17β-estradiol (E2), E2 + progesterone and E2 + BC (5 and 10 μM) and the effect of BC and progesterone on E2-induced cell proliferation was analyzed. BC decreased the proliferation of Ishikawa cells at a dose-dependent rate compared with the control group (p < 0.05). The proliferation of Ishikawa cells increased in the presence of E2, whereas the subsequent addition of progesterone or BC decreased proliferation to the level of the control group (p < 0.05). The inhibitory effect of BC on E2-induced cell proliferation was greater than the inhibitory effect of progesterone. In conclusion, BC induces apoptosis in Ishikawa cells and suppresses E2-induced cell proliferation in Ishikawa cells. BC could be considered a candidate co-treatment agent of estrogen-dependent tumors, especially those involving endometrial cells.

  17. Effects of 17 beta-estradiol on radiation transformation in vitro; inhibition of effects by protease inhibitors

    International Nuclear Information System (INIS)

    Kennedy, A.R.; Weichselbaum, R.R.

    1981-01-01

    We have investigated the effects of 17 beta-estradiol, given both alone and with X-irradiation, on the induction of malignant transformation in vitro. Treatment with 10(-6)M 17 beta-estradiol for 6 weeks, or 10(-5)M 17 beta-estradiol for only 5 days, induced malignant transformation in C3H 10T1/2 cells. Estradiol also acted as a cocarcinogen for X-ray induced transformation; the results indicate an additive effect when the cells were exposed to both agents together. The protease inhibitors antipain and leupeptin suppressed estradiol induced transformation as well as the additive effect observed for estradiol-radiation transformation

  18. Effects of 17 beta-estradiol on radiation transformation in vitro; inhibition of effects by protease inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Kennedy, A.R.; Weichselbaum, R.R.

    1981-01-01

    We have investigated the effects of 17 beta-estradiol, given both alone and with X-irradiation, on the induction of malignant transformation in vitro. Treatment with 10(-6)M 17 beta-estradiol for 6 weeks, or 10(-5)M 17 beta-estradiol for only 5 days, induced malignant transformation in C3H 10T1/2 cells. Estradiol also acted as a cocarcinogen for X-ray induced transformation; the results indicate an additive effect when the cells were exposed to both agents together. The protease inhibitors antipain and leupeptin suppressed estradiol induced transformation as well as the additive effect observed for estradiol-radiation transformation.

  19. Delta(9)-tetrahydrocannabinol inhibits 17beta-estradiol-induced proliferation and fails to activate androgen and estrogen receptors in MCF7 human breast cancer cells.

    Science.gov (United States)

    von Bueren, A O; Schlumpf, M; Lichtensteiger, W

    2008-01-01

    Delta(9)-tetrahydrocannabinol (THC) exerts palliative effects in cancer patients, but produces adverse effects on the endocrine and reproductive systems. Experimental evidence concerning such effects is controversial. Whether THC exhibits estrogenic or androgenic activity in vitro was investigated. Estrogenic effects of THC were analyzed in vitro by measuring the proliferation of estrogen-sensitive MCF7 cells. Androgenic activity was investigated by the A-Screen assay that measures androgen-dependent inhibition of proliferation of the androgen receptor (AR)-positive human mammary carcinoma cell line, MCF7-AR1. In contrast to 17beta-estradiol, included as positive control with an EC50 value (concentration required for 50% of maximal 17beta-estradiol-induced proliferation) of 1.00 x 10(-12) M, THC failed to induce cell proliferation in the MCF7 cell line at concentrations between 10(-13) and 10(-4) M. THC inhibited 17beta-estradiol-induced proliferation in wild-type MCF7 and MCF7-AR1 cells, with an IC50 value of 2.6 x 10(-5) M and 9 x 10(-6) M, respectively. THC failed to act as an estrogen, but antagonized 17beta-estradiol-induced proliferation. This effect was independent of the AR expression level.

  20. Estrogen receptor β inhibits estradiol-induced proliferation and migration of MCF-7 cells through regulation of mitofusin 2.

    Science.gov (United States)

    Ma, Li; Liu, Yueping; Geng, Cuizhi; Qi, Xiaowei; Jiang, Jun

    2013-06-01

    In the present study, we investigated whether estrogen receptor (ER) β affected the proliferation and migration of the human breast cancer cell line MCF-7 through regulation of mitofusin 2 (mfn2). A previous study reported that mfn2 may be regulated by ER through a non-classical pathway; in this pathway, the ER modulates the activities of other transcription factors by stabilizing their binding to DNA and/or recruiting coactivators to the complex. However, the previous study, unlike the study presented here, did not directly explore the interactions between ER and mfn2. Here, RT-PCR and western blot analysis were used to test the expression of mfn2 in MCF-7 cells after exposure to different doses of estradiol (E2). The ability of cells to proliferate and migrate was determined by MTT assay and a monolayer-wounding protocol, respectively. Finally, changes in MCF-7 cell biology after transfection with ERβ or mfn2 expression vectors were investigated, and the role of ERβ in mfn2 expression was also explored. Our results showed that E2 attenuated mfn2 expression in a dose-dependent manner, concomitant with the activation of proliferation and migration of MCF-7 cells. The mfn2 expression vector effectively suppressed E2-induced upregulation of PCNA and migration in MCF-7 cells. ERβ inhibited the E2-induced mfn2 downregulation that accompanied the inhibition of proliferation and migration in MCF-7 cells. Briefly, ERβ may inhibit E2-induced proliferation and migration of MCF-7 cells through regulation of mfn2.

  1. Estradiol modulates functional brain organization during the menstrual cycle : an analysis of interhemispheric inhibition.

    OpenAIRE

    Weis, S.; Hausmann, M.; Stoffers, B.; Vohn, R.; Kellermann, T.; Sturm, W.

    2008-01-01

    According to the hypothesis of progesterone-mediated interhemispheric decoupling (Hausmann and Güntürkün, 2000), functional cerebral asymmetries (FCAs), which are stable in men and change during the menstrual cycle in women, are generated by interhemispheric inhibition of the dominant on the nondominant hemisphere. The change of lateralization during the menstrual cycle in women might indicate that sex hormones play an important role in modulating FCAs. We used functional magnetic resonance i...

  2. Innate immunity in the vagina (part I): estradiol inhibits HBD2 and elafin secretion by human vaginal epithelial cells.

    Science.gov (United States)

    Patel, Mickey V; Fahey, John V; Rossoll, Richard M; Wira, Charles R

    2013-05-01

    Vaginal epithelial cells (VEC) are the first line of defense against incoming pathogens in the female reproductive tract. Their ability to produce the anti-HIV molecules elafin and HBD2 under hormonal stimulation is unknown. Vaginal epithelial cells were recovered using a menstrual cup and cultured overnight prior to treatment with estradiol (E₂), progesterone (P₄) or a panel of selective estrogen response modulators (SERMs). Conditioned media were recovered and analyzed for protein concentration and anti-HIV activity. E₂ significantly decreased the secretion of HBD2 and elafin by VEC over 48 hrs, while P4 and the SERMs (tamoxifen, PHTTP, ICI or Y134) had no effect. VEC conditioned media from E₂ -treated cells had no anti-HIV activity, while that from E₂ /P₄ -treated cells significantly inhibited HIV-BaL infection. The menstrual cup allows for effective recovery of primary VEC. Their production of HBD2 and elafin is sensitive to E₂, suggesting that innate immune protection varies in the vagina across the menstrual cycle. © 2013 John Wiley & Sons A/S.

  3. Knockdown of XBP1 by RNAi in Mouse Granulosa Cells Promotes Apoptosis, Inhibits Cell Cycle, and Decreases Estradiol Synthesis

    Directory of Open Access Journals (Sweden)

    Nan Wang

    2017-05-01

    Full Text Available Granulosa cells are crucial for follicular growth, development, and follicular atresia. X-box binding protein 1 (XBP1, a basic region-leucine zipper protein, is widely involved in cell differentiation, proliferation, apoptosis, cellular stress response, and other signaling pathways. In this study, RNA interference, flow cytometry, western blot, real-time PCR, Cell Counting Kit (CCK8, and ELISA were used to investigate the effect of XBP1 on steroidogenesis, apoptosis, cell cycle, and proliferation of mouse granulosa cells. ELISA analysis showed that XBP1 depletion significantly decreased the concentrations of estradiol (E2. Additionally, the expression of estrogen synthesis enzyme Cyp19a1 was sharply downregulated. Moreover, flow cytometry showed that knockdown of XBP1 increased the apoptosis rate and arrests the cell cycle in S-phase in granulosa cells (GCs. Further study confirmed these results. The expression of CCAAT-enhancer-binding protein homologous protein (CHOP, cysteinyl aspartate specific proteases-3 (caspase-3, cleaved caspase-3, and Cyclin E was upregulated, while that of Bcl-2, Cyclin A1, and Cyclin B1 was downregulated. Simultaneously, CCK8 analysis indicated that XBP1 disruption inhibited cell proliferation. In addition, XBP1 knockdown also alters the expression of Has2 and Ptgs2, two essential genes for folliculogenesis. Collectively, these data reveal a novel critical role of XBP1 in folliculogenesis by regulating the cell cycle, apoptosis, and steroid synthesis of mouse granulosa cells.

  4. Quantitative Structure-Activity Relationships Predicting the Antioxidant Potency of 17β-Estradiol-Related Polycyclic Phenols to Inhibit Lipid Peroxidation

    Directory of Open Access Journals (Sweden)

    Katalin Prokai-Tatrai

    2013-01-01

    Full Text Available The antioxidant potency of 17β-estradiol and related polycyclic phenols has been well established. This property is an important component of the complex events by which these types of agents are capable to protect neurons against the detrimental consequences of oxidative stress. In order to relate their molecular structure and properties with their capacity to inhibit lipid peroxidation, a marker of oxidative stress, quantitative structure-activity relationship (QSAR studies were conducted. The inhibition of Fe3+-induced lipid peroxidation in rat brain homogenate, measured through an assay detecting thiobarbituric acid reactive substances for about seventy compounds were correlated with various molecular descriptors. We found that lipophilicity (modeled by the logarithm of the n-octanol/water partition coefficient, logP was the property that influenced most profoundly the potency of these compounds to inhibit lipid peroxidation in the biological medium studied. Additionally, the important contribution of the bond dissociation enthalpy of the phenolic O-H group, a shape index, the solvent-accessible surface area and the energy required to remove an electron from the highest occupied molecular orbital were also confirmed. Several QSAR equations were validated as potentially useful exploratory tools for identifying or designing novel phenolic antioxidants incorporating the structural backbone of 17β-estradiol to assist therapy development against oxidative stress-associated neurodegeneration.

  5. Metformin inhibits 17β-estradiol-induced epithelial-to-mesenchymal transition via βKlotho-related ERK1/2 signaling and AMPKα signaling in endometrial adenocarcinoma cells.

    Science.gov (United States)

    Liu, Zhao; Qi, Shasha; Zhao, Xingbo; Li, Mingjiang; Ding, Sentai; Lu, Jiaju; Zhang, Hui

    2016-04-19

    The potential role of metformin in treating endometrial cancer remains to be explored. The current study investigated the role of metformin in 17β-estradiol-induced epithelial-mesenchymal transition (EMT) in endometrial adenocarcinoma cells. We found that 17β-estradiol promoted proliferation and migration, attenuated apoptosis in both estrogen receptor (ER) positive and ER negative endometrial adenocarcinoma cells (Ishikawa and KLE cells, respectively). Metformin abolished 17β-estradiol-induced cell proliferation and reversed 17β-estradiol-induced EMT in Ishikawa cells. In addition, metformin increased the expression of βKlotho, a fibroblast growth factors (FGFs) coreceptor, and decreased ERK1/2 phosphorylation in both Ishikawa and KLE cells. Decreased expression of βKlotho was noted in human endometrial adenocarcinomas, and plasmid-driven expression of βKlotho in Ishikawa cells abolished 17β-estradiol-induced EMT via inhibiting ERK1/2 signaling. βKlotho expression and metformin show synergetic effects on the proliferation and the EMT in Ishikawa cells. Furthermore, we demonstrated that the anti-EMT effects of metformin could be partly abolished by introducing Compound C, a specific AMPKα signaling inhibitor. In conclusion, metformin abolishes 17β-estradiol-induced cell proliferation and EMT in endometrial adenocarcinoma cells by upregulating βKlotho expression, inhibiting ERK1/2 signaling, and activating AMPKα signaling. Our study provides novel mechanistic insight into the anti-tumor effects of metformin.

  6. Chondrogenic properties of collagen type XI, a component of cartilage extracellular matrix.

    Science.gov (United States)

    Li, Ang; Wei, Yiyong; Hung, Clark; Vunjak-Novakovic, Gordana

    2018-08-01

    Cartilage extracellular matrix (ECM) has been used for promoting tissue engineering. However, the exact effects of ECM on chondrogenesis and the acting mechanisms are not well understood. In this study, we investigated the chondrogenic effects of cartilage ECM on human mesenchymal stem cells (MSCs) and identified the contributing molecular components. To this end, a preparation of articular cartilage ECM was supplemented to pellets of chondrogenically differentiating MSCs, pellets of human chondrocytes, and bovine articular cartilage explants to evaluate the effects on cell proliferation and the production of cartilaginous matrix. Selective enzymatic digestion and screening of ECM components were conducted to identify matrix molecules with chondrogenic properties. Cartilage ECM promoted MSC proliferation, production of cartilaginous matrix, and maturity of chondrogenic differentiation, and inhibited the hypertrophic differentiation of MSC-derived chondrocytes. Selective digestion of ECM components revealed a contributory role of collagens in promoting chondrogenesis. The screening of various collagen subtypes revealed strong chondrogenic effect of collagen type XI. Finally, collagen XI was found to promote production and inhibit degradation of cartilage matrix in human articular chondrocyte pellets and bovine articular cartilage explants. Our results indicate that cartilage ECM promotes chondrogenesis and inhibits hypertrophic differentiation in MSCs. Collagen type XI is the ECM component that has the strongest effects on enhancing the production and inhibiting the degradation of cartilage matrix. Copyright © 2018 Elsevier Ltd. All rights reserved.

  7. 17-beta estradiol inhibits oxidative stress-induced accumulation of AIF into nucleolus and PARP1-dependent cell death via estrogen receptor alpha.

    Science.gov (United States)

    Batnasan, Enkhzaya; Wang, Ruoxi; Wen, Jitao; Ke, Yueshuang; Li, Xiaoxue; Bohio, Ameer Ali; Zeng, Xianlu; Huo, Hongliang; Han, Liping; Boldogh, Istvan; Ba, Xueqing

    2015-01-05

    Oxidative stress-induced DNA damage results in over-activation of poly(ADP-ribose) polymerase 1 (PARP1), leading to parthanatos, a newly discovered cell elimination pathway. Inhibition of PARP1-dependent cell death has shown to improve the outcome of diseases, including stroke, heart ischemia, and neurodegenerative diseases. In the present study we aimed to detect whether estrogen plays a protective role in inhibiting parthanatos. We utilized human mammary adenocarcinoma cells (MCF7) that abundantly express the estrogen receptor alpha and beta (ERα and ERβ). Parthanatos was induced by challenging the cells with hydrogen peroxide (H2O2). Microscopic imaging and molecular biological techniques, such as Western blot analysis and RNA interference, were performed. The results showed 17β estradiol (E2) protected MCF7 cells from PARP1-dependent cell death by decreasing protein PARylation, and AIF translocation into nuclei/nucleoli. Down-regulation of ERα expression by siRNA before E2 addition resulted in the failure of the E2-mediated inhibition of H2O2-induced protein PARylation and AIF nucleolar translocation. Together these data suggest that estrogen via its alpha-type receptor inhibits oxidative stress-induced, PARP1-dependent cell death. The present study provided us insight into how to apply hormone therapy in intervention of parthanatos-implicated ischemic and degenerative diseases. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  8. Inhibition of the human liver microsomal and human cytochrome P450 1A2 and 3A4 metabolism of estradiol by deployment-related and other chemicals.

    Science.gov (United States)

    Usmani, Khawja A; Cho, Taehyeon M; Rose, Randy L; Hodgson, Ernest

    2006-09-01

    Cytochromes P450 (P450s) are major catalysts in the metabolism of xenobiotics and endogenous substrates such as estradiol (E2). It has previously been shown that E2 is predominantly metabolized in humans by CYP1A2 and CYP3A4 with 2-hydroxyestradiol (2-OHE2) the major metabolite. This study examines effects of deployment-related and other chemicals on E2 metabolism by human liver microsomes (HLM) and individual P450 isoforms. Kinetic studies using HLM, CYP3A4, and CYP1A2 showed similar affinities (Km) for E2 with respect to 2-OHE2 production. Vmax and CLint values for HLM are 0.32 nmol/min/mg protein and 7.5 microl/min/mg protein; those for CYP3A4 are 6.9 nmol/min/nmol P450 and 291 microl/min/nmol P450; and those for CYP1A2 are 17.4 nmol/min/nmol P450 and 633 microl/min/nmol P450. Phenotyped HLM use showed that individuals with high levels of CYP1A2 and CYP3A4 have the greatest potential to metabolize E2. Preincubation of HLM with a variety of chemicals, including those used in military deployments, resulted in varying levels of inhibition of E2 metabolism. The greatest inhibition was observed with organophosphorus compounds, including chlorpyrifos and fonofos, with up to 80% inhibition for 2-OHE2 production. Carbaryl, a carbamate pesticide, and naphthalene, a jet fuel component, inhibited ca. 40% of E2 metabolism. Preincubation of CYP1A2 with chlorpyrifos, fonofos, carbaryl, or naphthalene resulted in 96, 59, 84, and 87% inhibition of E2 metabolism, respectively. Preincubation of CYP3A4 with chlorpyrifos, fonofos, deltamethrin, or permethrin resulted in 94, 87, 58, and 37% inhibition of E2 metabolism. Chlorpyrifos inhibition of E2 metabolism is shown to be irreversible.

  9. 3,4-Dihydroxybenzaldehyde Derived from Prunus mume Seed Inhibits Oxidative Stress and Enhances Estradiol Secretion in Human Ovarian Granulosa Tumor Cells

    International Nuclear Information System (INIS)

    Kono, Ryohei; Nomura, Sachiko; Okuno, Yoshiharu; Nakamura, Misa; Maeno, Akihiro; Kagiya, Tomoko; Tokuda, Akihiko; Inada, Ken-ichi; Matsuno, Akira; Utsunomiya, Tomoko; Utsunomiya, Hirotoshi

    2014-01-01

    Granulosa cells form ovarian follicles and play important roles in the growth and maturation of oocytes. The protection of granulosa cells from cellular injury caused by oxidative stress is an effective therapy for female infertility. We here investigated an effective bioactive compound derived from Prunus mume seed extract that protects granulosa cells from hydrogen peroxide (H 2 O 2 )-induced apoptosis. We detected the bioactive compound, 3,4-dihydroxybenzaldehyde (3,4-DHBA), via bioactivity-guided isolation and found that it inhibited the H 2 O 2 -induced apoptosis of granulosa cells. We also showed that 3,4-DHBA promoted estradiol secretion in granulosa cells and enhanced the mRNA expression levels of steroidogenic factor 1, a promoter of key steroidogenic enzymes. These results suggest that P. mume seed extract may have clinical potential for the prevention and treatment of female infertility

  10. A cyclic peptide derived from alpha-fetoprotein inhibits the proliferative effects of the epidermal growth factor and estradiol in MCF7 cells.

    Science.gov (United States)

    Torres, Cristian; Antileo, Elmer; Epuñán, Maráa José; Pino, Ana María; Valladares, Luis Emilio; Sierralta, Walter Daniel

    2008-06-01

    A cyclic peptide derived from the active domain of alpha-fetoprotein (AFP) significantly inhibited the proliferation of MCF7 cells stimulated with the epidermal growth factor (EGF) or estradiol (E2). The action of these three agents on cell growth was independent of the presence of calf serum in the culture medium. Our results demonstrated that the cyclic peptide interfered markedly with the regulation of MAPK by activated c-erbB2. The cyclic peptide showed no effect on the E2-stimulated release of matrix metalloproteinases 2 and 9 nor on the shedding of heparin-binding EGF into the culture medium. We propose that the AFP-derived cyclic peptide represents a valuable novel antiproliferative agent for treating breast cancer.

  11. Mangiferin Reduces the Inhibition of Chondrogenic Differentiation by IL-1β in Mesenchymal Stem Cells from Subchondral Bone and Targets Multiple Aspects of the Smad and SOX9 Pathways

    Directory of Open Access Journals (Sweden)

    Jeong-Eun Huh

    2014-09-01

    Full Text Available Mangiferin is a natural immunomodulator found in plants including mango trees. The effects of mangiferin on chondrogenesis and cartilage repair have not yet been reported. This study was designed to determine the effect of mangiferin on chondrogenic differentiation in IL-1β-stimulated mesenchymal stem cells (MSCs from subchondral bone and to explore the mechanisms underlying these effects. MSCs were isolated from the subchondral bone of rabbit and treated with mangiferin alone and/or interleukin-1β (IL-1β. Mangiferin induced chondrogenic differentiation in MSCs by upregulating transforming growth factor (TGF-β, bone morphogenetic protein (BMP-2, and BMP-4 and several key markers of chondrogenesis, including sex-determining region Y–box (SRY-box containing gene 9 (SOX9, type 2α1 collagen (Col2α1, cartilage link protein, and aggrecan. In IL-1β-stimulated MSCs, mangiferin significantly reversed the production of TGF-β, BMP-2, BMP-4, SOX9, Col2α1, cartilage link protein, and aggrecan, as well as matrix metalloproteinase (MMP-1, MMP-13, and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS5. Mangiferin upregulated the phosphorylation of Smad 2, Smad 3, Smad 1/5/8, and SOX9 in IL-1β-stimulated MSCs. In the presence of mangiferin, SOX9 siRNA suppressed the activation of Smad 2, Smad 3, Smad 1/5/8, aggrecan, and Col2α1 expression. In conclusion, mangiferin exhibits both chondrogenic and chondroprotective effects on damaged MSCs and mediates these effects by targeting multiple aspects of the Smad and SOX9 signaling pathways.

  12. Acute inhibition of estradiol synthesis impacts vestibulo-ocular reflex adaptation and cerebellar long-term potentiation in male rats.

    Science.gov (United States)

    Dieni, Cristina V; Ferraresi, Aldo; Sullivan, Jacqueline A; Grassi, Sivarosa; Pettorossi, Vito E; Panichi, Roberto

    2018-03-01

    The vestibulo-ocular reflex (VOR) adaptation is an ideal model for investigating how the neurosteroid 17 beta-estradiol (E2) contributes to the modification of behavior by regulating synaptic activities. We hypothesized that E2 impacts VOR adaptation by affecting cerebellar synaptic plasticity at the parallel fiber-Purkinje cell (PF) synapse. To verify this hypothesis, we investigated the acute effect of blocking E2 synthesis on gain increases and decreases in adaptation of the VOR in male rats using an oral dose (2.5 mg/kg) of the aromatase inhibitor letrozole. We also assessed the effect of letrozole on synaptic plasticity at the PF synapse in vitro, using cerebellar slices from male rats. We found that letrozole acutely impaired both gain increases and decreases adaptation of the VOR without altering basal ocular-motor performance. Moreover, letrozole prevented long-term potentiation at the PF synapse (PF-LTP) without affecting long-term depression (PF-LTD). Thus, in male rats neurosteroid E2 has a relevant impact on VOR adaptation and affects exclusively PF-LTP. These findings suggest that E2 might regulate changes in VOR adaptation by acting locally on cerebellar and extra-cerebellar synaptic plasticity sites.

  13. Methylseleninic acid (MSA) inhibits 17β-estradiol-induced cell growth in breast cancer T47D cells via enhancement of the antioxidative thioredoxin/ thioredoxin reductase system.

    Science.gov (United States)

    Okuno, Tomofumi; Miura, Kiyoshi; Sakazaki, Fumitoshi; Nakamuro, Katsuhiko; Ueno, Hitoshi

    2012-01-01

    The purpose of this study was to clarify the cell growth inhibitory mechanism of human breast cancer cells caused by selenium (Se) compounds. In the presence of 17β-estradiol (E(2)) at physiological concentrations, growth of estrogen receptor α (ERα)-positive T47D cells was markedly inhibited by 1 × 10(-6) mol/L methylseleninic acid (MSA) with no Se related toxicity.Under conditions where cell growth was inhibited, MSA decreased ERα mRNA levels and subsequent protein levels; further decreasing expression of estrogen-responsive finger protein (Efp) which is a target gene product of ERα and promotes G2/M progression of the cell cycle. Therefore, the decline in Efp expression is presumed to be involved in G2 arrest. Coincidentally, the antioxidative thioredoxin/ thioredoxin reductase (Trx/TrxR) system in cells was enhanced by the synergistic action of E(2) and MSA. It has been reported that ROS-induced oxidative stress enhanced ERα expression. E(2) increased production of intracellular ROS in T47D cells. Meanwhile, MSA significantly decreased E(2)-induced ROS accumulation. From these results, activation of the Trx/TrxR system induced by the coexistence of MSA and E(2) suppresses oxidative stress and decreases expression of ERα, and finally induces the growth arrest of T47D cells through disruption of ERα signaling.

  14. Metformin inhibits 17?-estradiol-induced epithelial-to-mesenchymal transition via ?Klotho-related ERK1/2 signaling and AMPK? signaling in endometrial adenocarcinoma cells

    OpenAIRE

    Liu, Zhao; Qi, Shasha; Zhao, Xingbo; Li, Mingjiang; Ding, Sentai; Lu, Jiaju; Zhang, Hui

    2016-01-01

    The potential role of metformin in treating endometrial cancer remains to be explored. The current study investigated the role of metformin in 17?-estradiol-induced epithelial-mesenchymal transition (EMT) in endometrial adenocarcinoma cells. We found that 17?-estradiol promoted proliferation and migration, attenuated apoptosis in both estrogen receptor (ER) positive and ER negative endometrial adenocarcinoma cells (Ishikawa and KLE cells, respectively). Metformin abolished 17?-estradiol-induc...

  15. Endocrine control of sexual behavior in sneaker males of the peacock blenny Salaria pavo: effects of castration, aromatase inhibition, testosterone and estradiol.

    Science.gov (United States)

    Gonçalves, David; Alpedrinha, João; Teles, Magda; Oliveira, Rui F

    2007-04-01

    The effects of castration and sex steroid manipulations on the expression of sexual behavior were investigated in a small fish, the peacock blenny, Salaria pavo. In this species, large males defend nests and attract females while small "sneaker" males reproduce by imitating the female morphology and courtship behavior in order to approach nests during spawning events and parasitically fertilize eggs. Sneakers switch into nest holders in their second breeding season, thus displaying both male and female-like sexual behavior during their lifetime. We tested the effects of castration and of an aromatase inhibitor (Fadrozole, F), testosterone (T) or 17beta-estradiol (E(2)) implants on the expression of male and female-like behavior in sneakers. Sneakers were either sham-operated, castrated or castrated and implanted with vehicle, F, T+F or E(2)+F. Seven days after the treatment, sneakers were placed in a tank with a nesting male, two ripe females and an available nest. Castrated fish had lower levels of circulating T and increased the time spent displaying female typical nuptial coloration. T implants had the opposite effect, inhibiting the expression of female-like behavior and coloration. E(2) implants had no significant effect on the display of sexual behavior but the frequency of aggressive displays decreased. The results agree with previous findings in sneakers of S. pavo that demonstrated an inhibition of female-like behavior by 11-ketotestosterone (11-KT). The reported increase in T and 11-KT production when sneakers change into nest holders may thus contribute to behaviorally defeminize sneakers. Contrarily, both T and E(2) failed to promote male-like behavior, suggesting that behavioral masculization during tactic switching depends on other neuroendocrine mechanisms or that the time length of the experiment was insufficient to induce male-like behavioral changes in sneakers.

  16. 17beta-estradiol induced vitellogenesis is inhibited by cortisol at the post-transcriptional level in Arctic char (Salvelinus alpinus

    Directory of Open Access Journals (Sweden)

    Modig Carina

    2004-09-01

    Full Text Available Abstract This study was performed to investigate stress effects on the synthesis of egg yolk precursor, vitellogenin (Vtg in Arctic char (Salvelinus alpinus. In particular the effect of cortisol (F was determined since this stress hormone has been suggested to interfere with vitellogenesis and is upregulated during sexual maturation in teleosts. Arctic char Vtg was purified and polyclonal antibodies were produced in order to develop tools to study regulation of vitellogenesis. The Vtg antibodies were used to develop an enzyme-linked immunosorbent assay. The corresponding Vtg cDNA was cloned from a hepatic cDNA library in order to obtain DNA probes to measure Vtg mRNA expression. Analysis of plasma from juvenile Arctic char, of both sexes, exposed to different steroids showed that production of Vtg was induced in a dose dependent fashion by 17β-estradiol (E2, estrone and estriol. Apart from estrogens a high dose of F also upregulated Vtg. In addition, F, progesterone (P and tamoxifen were tested to determine these compounds ability to modulate E2 induced Vtg synthesis at both the mRNA and protein level. Tamoxifen was found to inhibit E2 induced Vtg mRNA and protein upregulation. P did not alter the Vtg induction while F reduced the Vtg protein levels without affecting the Vtg mRNA levels. Furthermore the inhibition of Vtg protein was found to be dose dependent. Thus, the inhibitory effect of F on Vtg appears to be mediated at the post-transcriptional level.

  17. TIS21/(BTG2) negatively regulates estradiol-stimulated expansion of hematopoietic stem cells by derepressing Akt phosphorylation and inhibiting mTOR signal transduction.

    Science.gov (United States)

    Kim, Bong Cho; Ryu, Min Sook; Oh, S Paul; Lim, In Kyoung

    2008-09-01

    It has been known that 12-O-tetradecanoyl phorbol-13-acetate-inducible sequence 21 (TIS21), ortholog of human B-cell translocation gene 2, regulates expansions of stage-specific thymocytes and hematopoietic progenitors. In the present study, lineage-negative (Lin(-))/stem cell antigen-1-positive (Sca-1+)/c-Kit+ (LSK) cell content was significantly elevated in bone marrow (BM) of TIS21-knockout (TIS21(-/-)) female mice, suggesting 17beta-estradiol (E(2))-regulated progenitor expansion. E(2) induced DNA synthesis and cell proliferation of mouse embryonic fibroblasts (MEFs) isolated from TIS21(-/-) mice, but not wild type (WT). In contrast to WT, E(2) failed to activate protein kinase B (Akt) in the TIS21(-/-) MEFs, independent of extracellular signal-regulated kinase 1/2 (Erk1/2) activation. Despite attenuation of Akt activation, mammalian target of rapamycin (mTOR) was constitutively activated in the TIS21(-/-) MEFs. Furthermore, mitogen-activated protein kinase 1/2 inhibitor or knockdown of Erk1 could restore activation of Akt and downregulate mTOR. Immunoprecipitation showed Akt preferentially bound to phosphorylated Erk1/2 (p-Erk1/2) in TIS21(-/-) cells, but reconstitution of TIS21 inhibited their interaction. E(2)-injected TIS21(-/-) male mice also increased LSK cells in BM. Taken together, expansion of hematopoietic progenitors in TIS21(-/-) female mice might be through inhibition of Akt activation, and constitutive activation of mTOR via preferential binding of TIS21 to E(2)-induced p-Erk1/2, compared with that of Akt. Our results suggest that TIS21 plays a pivotal role in maintaining the hematopoietic stem cell compartment and hematopoiesis.

  18. Expressions of pathologic markers in PRP based chondrogenic differentiation of human adipose derived stem cells.

    Science.gov (United States)

    Pakfar, Arezou; Irani, Shiva; Hanaee-Ahvaz, Hana

    2017-02-01

    -based chondrogenic differentiation media is required to inhibit the production of angiogenic/inflammatory markers, calcification and the release of synthesized GAG out of the construct. Copyright © 2016. Published by Elsevier Ltd.

  19. Blood Test: Estradiol

    Science.gov (United States)

    ... the bloodstream. Estradiol plays an important role in sexual development: It's the most important form of the hormone ... while low levels may indicate a delay in sexual development. Estradiol levels also give important information on the ...

  20. Lipoxin A4 regulates expression of the estrogen receptor and inhibits 17β-estradiol induced p38 mitogen-activated protein kinase phosphorylation in human endometriotic stromal cells.

    Science.gov (United States)

    Chen, Shuo; Wu, Rong-Feng; Su, Lin; Zhou, Wei-Dong; Zhu, Mao-Bi; Chen, Qiong-Hua

    2014-07-01

    To study the role of lipoxin A4 (LXA4) in endometriosis. Molecular analysis in human samples and primary human endometriotic stromal cells (ESCs). University hospital. Forty-nine premenopausal women (30 patients with endometriosis and 19 controls). Normal and ectopic endometrial biopsies obtained during surgery performed during the proliferative phase of the menstrual cycle; ESCs used for in vitro studies. Levels of LXA4 measured by enzyme-linked immunosorbent assay (ELISA); mRNA levels of the estrogen receptor (ER), progestogen receptor (PR), tumor necrosis factor α (TNF-α), and interleukin 6 (IL-6) quantified by quantitative reverse-transcription polymerase chain reaction (qRT-PCR); and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation evaluated by Western blotting. The LXA4 expression level decreased in ectopic tissue as well as ERα and PR, although the expression of ERβ increased in ectopic endometrium compared with the controls. Investigations with correlation analysis revealed the expression of LXA4 was positively correlated with ERα and negatively correlated with ERβ in vivo. Moreover, administering LXA4 could augment ERβ expression in ESCs and inhibit the 17β-estradiol-induced phosphorylation of p38 MAPK very likely through ERβ. Our findings indicate that LXA4 regulates ERβ expression and inhibits 17β-estradiol-induced phosphorylation of p38 MAPK, very likely through ERβ in ESCs. Copyright © 2014. Published by Elsevier Inc.

  1. Estradiol induces region-specific inhibition of ZENK but does not affect the behavioral preference for tutored song in adult female zebra finches

    OpenAIRE

    Svec, Lace A.; Wade, Juli

    2008-01-01

    Female zebra finches display a preference for songs of males raised with tutors compared to those from males without tutors. To determine howthis behavioral preference may bemediated by auditory perception sites, the social behavior network, and the dopamine reward system, and whether responses of these regions are affected by estradiol, females were treated with hormone or blank implants.An auditory choice test was conducted followed by exposure to tutored or untutored song or silence to exa...

  2. Polymodal Transient Receptor Potential Vanilloid (TRPV Ion Channels in Chondrogenic Cells

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    Csilla Szűcs Somogyi

    2015-08-01

    Full Text Available Mature and developing chondrocytes exist in a microenvironment where mechanical load, changes of temperature, osmolarity and acidic pH may influence cellular metabolism. Polymodal Transient Receptor Potential Vanilloid (TRPV receptors are environmental sensors mediating responses through activation of linked intracellular signalling pathways. In chondrogenic high density cultures established from limb buds of chicken and mouse embryos, we identified TRPV1, TRPV2, TRPV3, TRPV4 and TRPV6 mRNA expression with RT-PCR. In both cultures, a switch in the expression pattern of TRPVs was observed during cartilage formation. The inhibition of TRPVs with the non-selective calcium channel blocker ruthenium red diminished chondrogenesis and caused significant inhibition of proliferation. Incubating cell cultures at 41 °C elevated the expression of TRPV1, and increased cartilage matrix production. When chondrogenic cells were exposed to mechanical load at the time of their differentiation into matrix producing chondrocytes, we detected increased mRNA levels of TRPV3. Our results demonstrate that developing chondrocytes express a full palette of TRPV channels and the switch in the expression pattern suggests differentiation stage-dependent roles of TRPVs during cartilage formation. As TRPV1 and TRPV3 expression was altered by thermal and mechanical stimuli, respectively, these are candidate channels that contribute to the transduction of environmental stimuli in chondrogenic cells.

  3. Inverse Regulation of Early and Late Chondrogenic Differentiation by Oxygen Tension Provides Cues for Stem Cell-Based Cartilage Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Sophie Portron

    2015-01-01

    Full Text Available Background/Aims: Multipotent stem/stromal cells (MSC are considered promising for cartilage tissue engineering. However, chondrogenic differentiation of MSC can ultimately lead to the formation of hypertrophic chondrocytes responsible for the calcification of cartilage. To prevent the production of this calcified matrix at the articular site, the late hypertrophic differentiation of MSCs must be carefully controlled. Given that articular cartilage is avascular, we hypothesized that in addition to its stimulatory role in the early differentiation of chondrogenic cells, hypoxia may prevent their late hypertrophic conversion. Methods: Early and late chondrogenic differentiation were evaluated using human adipose MSC and murine ATDC5 cells cultured under either normoxic (21%O2 or hypoxic (5%O2 conditions. To investigate the effect of hypoxia on late chondrogenic differentiation, the transcriptional activity of hypoxia-inducible factor-1alpha (HIF-1α and HIF-2α were evaluated using the NoShift DNA-binding assay and through modulation of their activity (chemical inhibitor, RNA interference. Results: Our data demonstrate that low oxygen tension not only stimulates the early chondrogenic commitment of two complementary models of chondrogenic cells, but also inhibits their hypertrophic differentiation. Conclusion: These results suggest that hypoxia can be used as an instrumental tool to prevent the formation of a calcified matrix in MSC-based cartilage tissue engineering.

  4. SPRY4-mediated ERK1/2 signaling inhibition abolishes 17β-estradiol-induced cell growth in endometrial adenocarcinoma cell.

    Science.gov (United States)

    Li, Mingjiang; Zhang, Hui; Zhao, Xingbo; Yan, Lei; Wang, Chong; Li, Chunyan; Li, Changzhong

    2014-08-01

    Basic fibroblast growth factor (FGF2)-mediated Extracellular signal-regulated kinases1/2 (ERK1/2) signaling is a critical modulator in angiogenesis. SPRY4 has been reported to be a feedback negative regulator of FGFs-induced ERK1/2 signaling. The aim of this study was to explore the role of SPRY4 in endometrial adenocarcinoma cell. The effect of SPRY4 expression on FGF2-mediated ERK1/2 signaling was detected by luciferase assay and Western blot analysis. The growth of Ishikawa cells was detected using colony formation assay and cell number counting experiment. We found that plasmid-driven SPRY4 expression efficiently blocked the activity of FGF2-induced ERK1/2 signaling in Ishikawa cells. SPRY4 expression significantly reduced the proliferation and 17β-estradiol-induced proliferation of Ishikawa cells. SPRY4 may function as a tumor suppressor in endometrial adenocarcinoma.

  5. Estradiol Transdermal Patch

    Science.gov (United States)

    ... menopause (change of life; the end of monthly menstrual periods). Transdermal estradiol is also used to prevent ... patch. Ask your pharmacist or doctor for a copy of the manufacturer's information for the patient.

  6. Elevated levels of mitochonrial respiratory complexes activities and ATP production in 17-β-estradiol-induced prolactin-secretory tumor cells in male rats are inhibited by melatonin in vivo and in vitro.

    Science.gov (United States)

    Wang, Bao-Qiang; Yang, Quan-Hui; Xu, Rong-Kun; Xu, Jian-Ning

    2013-01-01

    Our earlier studies indicate that melatonin inhibits the proliferation of prolactinoma and induces apoptosis of pituitary prolactin-secreting tumor in rats. Melatonin has also been shown to induce apoptosis and to reduce the production of ATP in breast tumor cells. This study analyzed the levels of the four mitochondrial respiratory complexes and the production of ATP and also the effects of melatonin treatment of prolactinoma. In the in vivo study, mitochondria were harvested from control pituitaries or prolactinoma collected from the pituitaries of melatonin- and 17-β-estradiol (E2)-treated male rats. In the in vitro study, prolactinoma cells mitochondria were harvested. Activities of the four mitochondrial respiratory complexes were assayed using fluorometer. ATP production of prolactinoma cells was estimated using bioluminescent methods. Elevated levels of four mitochondrial respiratory complexes activities and ATP production were recorded in prolactinoma cells. Moreover, in both in vivo and in vitro studies, melatonin inhibited the activities of mitochondrial respiratory complexes and the production of ATP in prolactinoma cells. There is a link between mitochondrial function increase and tumorigenesis. Melatonin induces apoptosis of pituitary prolactin-secreting tumor of rats via the induction of mitochondrial dysfunction and inhibition of energy metabolism.

  7. Long-Term Expansion, Enhanced Chondrogenic Potential, and Suppression of Endochondral Ossification of Adult Human MSCs via WNT Signaling Modulation

    Directory of Open Access Journals (Sweden)

    Roberto Narcisi

    2015-03-01

    Full Text Available Mesenchymal stem cells (MSCs are a potential source of chondrogenic cells for the treatment of cartilage disorders, but loss of chondrogenic potential during in vitro expansion and the propensity of cartilage to undergo hypertrophic maturation impede their therapeutic application. Here we report that the signaling protein WNT3A, in combination with FGF2, supports long-term expansion of human bone marrow-derived MSCs. The cells retained their chondrogenic potential and other phenotypic and functional properties of multipotent MSCs, which were gradually lost in the absence of WNT3A. Moreover, we discovered that endogenous WNT signals are the main drivers of the hypertrophic maturation that follows chondrogenic differentiation. Inhibition of WNT signals during differentiation prevented calcification and maintained cartilage properties following implantation in a mouse model. By maintaining potency during expansion and preventing hypertrophic maturation following differentiation, the modulation of WNT signaling removes two major obstacles that impede the clinical application of MSCs in cartilage repair.

  8. A synthetic peptide derived from alpha-fetoprotein inhibits the estradiol-induced proliferation of mammary tumor cells in culture through the modulation of p21.

    Science.gov (United States)

    Sierralta, Walter D; Epuñan, María J; Reyes, José M; Valladares, Luis E; Pino, Ana M

    2008-01-01

    A stable cyclized 9-mer peptide (cP) containing the active site of alpha-alpha fetoprotein (alphaFP) has been shown to be effective for prevention of estrogen-stimulated tumor cell proliferation in culture or of xenographt growth in immunodeficient mice. cP does not block 17beta-estradiol (E2) binding to its receptors, but rather appears to interfere with intracellular processing of the signal that supports growth. To obtain insight on that mechanism we studied the effect of cP on the proliferation of MCF-7 cells in culture. Proliferation in the presence of 2 microM E2 is decreased up to 40% upon addition of 2 microg ml(-1) cP to the medium; the presence of cP did not increase cell death, cP reduced also the proliferation of estrogen-dependent ZR75-1 cells but had no effect on autonomous MDA-MB-231 cells, cP did not modify the number of binding sites for labeled E2 or affected cell death. We detected increased nuclear p21Cip1 immunoreactivity after cP treatment. Our results suggest that cP acts via p21Cip1 to slow the process of MCF-7 cells through the cycle.

  9. [Corrigendum] Estrogen receptor β inhibits estradiol-induced proliferation and migration of MCF-7 cells through regulation of mitofusin 2.

    Science.gov (United States)

    Ma, Li; Liu, Yueping; Geng, Cuizhi; Qi, Xiaowei; Jiang, Jun

    2016-11-01

    Following the publication of this article, an interested reader drew to our attention an anomaly associated with Fig. 2, which presented the results of a wound‑healing assay performed to determine whether estradiol (E2) was able to exert an influence on MCF‑7 cell migration. Specifically, in comparing the 'Control' and the '10‑8/mol/l' panels, the data for the 'Control' panel had erroneously been included as the '10‑8/mol/l' panel, albeit the latter panel appeared in a slightly reorientated position and was stretched longitudinally. After having re‑examined our original data, we realize that the inclusion of the identical data for the '10‑8/mol/l' panel was incorrect. Subsequently, we have re-captured the images, and a corrected version of Fig. 2 is presented here. The Figure now correctly demonstrates that E2 enhanced cell motility in a dose‑dependent manner in the concentration range of E2 from 10-9 mol/l to 10-6 mol/l. This error did not overall affect the conclusions reported in the study. We sincerely apologize for this mistake, and thank the reader of our article who drew this matter to our attention. Furthermore, we regret any inconvenience this mistake has caused. [the original article was published in the International Journal of Oncology 42: 1993‑2000, 2013; DOI: 10.3892/ijo.2013.1903].

  10. Chondrogenic potential of mesenchymal stem cells from patients with rheumatoid arthritis and osteoarthritis: measurements in a microculture system.

    Science.gov (United States)

    Dudics, Valeria; Kunstár, Aliz; Kovács, János; Lakatos, Tamás; Géher, Pál; Gömör, Béla; Monostori, Eva; Uher, Ferenc

    2009-01-01

    Mesenchymal stem cells (MSCs) have the potential to differentiate into distinct mesenchymal tissues; including cartilage and bone, they can be an attractive cell source for cartilage tissue engineering approaches. Our objective here was to compare the in vitro chondrogenic potential of MSCs isolated from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) with cells from normal donors. Marrow samples were removed during bone surgery and adherent cell cultures were established. The cells were then passed into a newly developed microaggregate culture system in a medium containing transforming growth factor beta3, insulin, dexamethasone and/or demineralized bone matrix. In vitro chondrogenic activity was measured as metabolic sulfate incorporation and type II collagen expression in pellet cultures. Culture-expanded MSCs from RA and OA patients did not differ significantly from the normal population with respect to their chondrogenic potential in vitro. Capability of total protein and proteoglycan synthesis as well as collagen II mRNA expression by cell aggregates was similar for all cell preparations in the presence of the appropriate growth and differentiation factors. Chondroprotective drugs such as chondroitin sulfate and glucosamine enhanced, whereas chloroquine inhibited chondrogenesis in normal donor-derived or patient-derived MSC cultures. Galectin-1, a beta-galactoside-binding protein with marked anti-inflammatory activity, stimulated the chondrogenic differentiation of mesenchymal cells in low (<2 microg/ml) concentration. These findings show that MSCs from RA and OA patients possess similar chondrogenic potential as MSCs isolated from healthy donors, therefore these cells may serve as a potential new prospect in cartilage replacement therapy. 2008 S. Karger AG, Basel.

  11. Progestins inhibit estradiol-induced vascular endothelial growth factor and stromal cell-derived factor 1 in human endometrial stromal cells.

    Science.gov (United States)

    Okada, Hidetaka; Okamoto, Rika; Tsuzuki, Tomoko; Tsuji, Shoko; Yasuda, Katsuhiko; Kanzaki, Hideharu

    2011-09-01

    To investigate whether 17β-estradiol (E(2)) and progestins exert direct effects on vascular endothelial growth factor (VEGF) and stromal cell-derived factor 1 (SDF-1/CXCL12) in human endometrial stromal cells (ESCs) and thereby to clarify the regulatory function of these local angiogenic factors in the endometrium. In vitro experiment. Research laboratory at Kansai Medical University. Fourteen patients undergoing hysterectomy for benign reasons. ESCs were cultured with E(2) and/or various clinically relevant progestins (medroxyprogesterone acetate [MPA], norethisterone [NET], levonorgestrel [LNG], dienogest [DNG], and progesterone [P]). The mRNA levels and production of VEGF and SDF-1 were assessed by real-time reverse-transcription polymerase chain reaction and ELISA, respectively. E(2) significantly induced the mRNA levels and protein production of VEGF and SDF-1 in ESCs. MPA could antagonize the E(2)-stimulated effects in a time- and dose-dependent manner, and this effect could be reversed by RU-486 (P receptor antagonist). All of the progestins (MPA, NET, LNG, and DNG; 10(-9) to 10(-7) mol/L) attenuated E(2)-induced VEGF and SDF-1 production, whereas P showed these inhibitory effects only when present in a high concentration (10(-7) mol/L). Progestins have inhibitory effects on E(2)-induced VEGF and SDF-1 in ESCs. These results may indicate a potential mechanism for action of the female sex steroids in the human endometrium that can be helpful for various clinical applications. Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  12. Anterior cruciate ligament-derived cells have high chondrogenic potential.

    Science.gov (United States)

    Furumatsu, Takayuki; Hachioji, Motomi; Saiga, Kenta; Takata, Naoki; Yokoyama, Yusuke; Ozaki, Toshifumi

    2010-01-01

    Anterior cruciate ligament (ACL)-derived cells have a character different from medial collateral ligament (MCL)-derived cells. However, the critical difference between ACL and MCL is still unclear in their healing potential and cellular response. The objective of this study was to investigate the mesenchymal differentiation property of each ligament-derived cell. Both ligament-derived cells differentiated into adipogenic, osteogenic, and chondrogenic lineages. In chondrogenesis, ACL-derived cells had the higher chondrogenic property than MCL-derived cells. The chondrogenic marker genes, Sox9 and alpha1(II) collagen (Col2a1), were induced faster in ACL-derived pellets than in MCL-derived pellets. Sox9 expression preceded the increase of Col2a1 in both pellet-cultured cells. However, the expression level of Sox9 and a ligament/tendon transcription factor Scleraxis did not parallel the increase of Col2a1 expression along with chondrogenic induction. The present study demonstrates that the balance between Sox9 and Scleraxis have an important role in the chondrogenic differentiation of ligament-derived cells. Copyright 2009 Elsevier Inc. All rights reserved.

  13. Estradiol blood test

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003711.htm Estradiol blood test To use the sharing features on this page, ... of estrogens. How the Test is Performed A blood sample is needed . How to Prepare for the Test Your health care provider may tell you to ...

  14. Cell type dependent morphological adaptation in polyelectrolyte hydrogels governs chondrogenic fate.

    Science.gov (United States)

    Raghothaman, Deepak; Leong, Meng Fatt; Lim, Tze Chiun; Wan, Andrew C A; Ser, Zheng; Lee, Eng Hin; Yang, Zheng

    2016-04-04

    Repair of critical-size articular cartilage defects typically involves delivery of cells in biodegradable, 3D matrices. Differences in the developmental status of mesenchymal stem cells (MSCs) and terminally differentiated mature chondrocytes might be a critical factor in engineering appropriate 3D matrices for articular cartilage tissue engineering. This study examined the relationship between material-driven early cell morphological adaptations and chondrogenic outcomes, by studying the influence of aligned collagen type I (Col I) presentation on chondrocytes and MSC in interfacial polyelectrolyte complexation (IPC)-based hydrogels. In the absence of Col I, both chondrocytes and MSCs adopted rounded cell morphology and formed clusters, with chondrocyte clusters favoring the maintenance of hyaline phenotype, while MSC clusters differentiated to fibro-superficial zone-like chondrocytes. Encapsulated chondrocytes in IPC-Col I hydrogel adopted a fibroblastic morphology forming fibro-superficial zone-like phenotype, which could be reversed by inhibiting actin polymerization using cytochalasin D (CytD). In contrast, adoption of fibroblastic morphology by encapsulated MSCs in IPC-Col I facilitated superior chondrogenesis, generating a mature, hyaline neocartilage tissue. CytD treatment abrogated the elongation of MSCs and brought about a single cell-like state, resulting in insignificant chondrogenic differentiation, underscoring the essential requirement of providing matrix environments that are amenable to cell-cell interactions for robust MSC chondrogenic differentiation. Our study demonstrates that MSCs and culture-expanded chondrocytes favour differential microenvironmental niches and emphasizes the importance of designing biomaterials that meet cell type-specific requirements, in adopting chondrocyte or MSC-based approaches for regenerating hyaline, articular cartilage.

  15. TrxR2 deficiencies promote chondrogenic differentiation and induce apoptosis of chondrocytes through mitochondrial reactive oxygen species

    International Nuclear Information System (INIS)

    Yan, Jidong; Xu, Jing; Fei, Yao; Jiang, Congshan; Zhu, Wenhua; Han, Yan; Lu, Shemin

    2016-01-01

    Thioredoxin reductase 2 (TrxR2) is a selenium (Se) containing protein. Se deficiency is associated with an endemic osteoarthropathy characterized by impaired cartilage formation. It is unclear whether TrxR2 have roles in cartilage function. We examined the effects of TrxR2 on chondrogenic ATDC5 cells through shRNA-mediated gene silencing of TrxR2. We demonstrated TrxR2 deficiencies could enhance chondrogenic differentiation and apoptosis of ATDC5 cells. TrxR2 deficiencies increased accumulation of cartilage glycosaminoglycans (GAGs) and mineralization. TrxR2 deficiencies also stimulated expression of extracellular (ECM) gene including Collagen II and Aggrecan. The enhanced chondrogenic properties were further confirmed by activation of Akt signaling which are required for chondrogenesis. In addition, TrxR2 deficiencies promoted chondrocyte proliferation through acceleration of cell cycle progression by increase in both S and G2/M phase cell distribution accompanied with induction of parathyroid hormone-related protein (PTHrP). Moreover, TrxR2 deficiencies induced chondrocyte death via apoptosis and increased cell sensitivity to exogenous oxidative stress. Furthermore, TrxR2 deficiencies induced emission of mitochondrial reactive oxygen species (ROS) without alteration of mitochondrial membrane potential and intracellular ATP content. Finally, treatment of TrxR2 deficiency cells with N-acetylcysteine (NAC) inhibited mitochondrial ROS production and chondrocyte apoptosis. NAC also prevented chondrogenic differentiation of TrxR2 deficiency cells by suppression of ECM gene expression, GAGs accumulation and mineralization, as well as attenuation of Akt signaling. Thus, TrxR2-mediated mitochondrial integrity is indispensable for chondrogenic differentiation of ATDC5 cells. TrxR2 deficiency-induced impaired proliferation and death of chondrocytes may be the pathological mechanism of the osteoarthropathy due to Se deficiency. Notably, this study also uncover the roles of

  16. TrxR2 deficiencies promote chondrogenic differentiation and induce apoptosis of chondrocytes through mitochondrial reactive oxygen species

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Jidong [Department of Human Anatomy, Histology and Embryology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, Xi’an, Shaanxi 710061 (China); Xu, Jing [Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, Xi’an, Shaanxi 710061 (China); Fei, Yao [College of Life Sciences, Northwest University, Xi’an, Shaanxi Province 710069 (China); Jiang, Congshan; Zhu, Wenhua; Han, Yan [Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, Xi’an, Shaanxi 710061 (China); Lu, Shemin, E-mail: lushemin@xjtu.edu.cn [Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, Xi’an, Shaanxi 710061 (China); Key Laboratory of Environment and Genes Related to Diseases, Xi’an Jiaotong University, Ministry of Education of China (China)

    2016-05-15

    Thioredoxin reductase 2 (TrxR2) is a selenium (Se) containing protein. Se deficiency is associated with an endemic osteoarthropathy characterized by impaired cartilage formation. It is unclear whether TrxR2 have roles in cartilage function. We examined the effects of TrxR2 on chondrogenic ATDC5 cells through shRNA-mediated gene silencing of TrxR2. We demonstrated TrxR2 deficiencies could enhance chondrogenic differentiation and apoptosis of ATDC5 cells. TrxR2 deficiencies increased accumulation of cartilage glycosaminoglycans (GAGs) and mineralization. TrxR2 deficiencies also stimulated expression of extracellular (ECM) gene including Collagen II and Aggrecan. The enhanced chondrogenic properties were further confirmed by activation of Akt signaling which are required for chondrogenesis. In addition, TrxR2 deficiencies promoted chondrocyte proliferation through acceleration of cell cycle progression by increase in both S and G2/M phase cell distribution accompanied with induction of parathyroid hormone-related protein (PTHrP). Moreover, TrxR2 deficiencies induced chondrocyte death via apoptosis and increased cell sensitivity to exogenous oxidative stress. Furthermore, TrxR2 deficiencies induced emission of mitochondrial reactive oxygen species (ROS) without alteration of mitochondrial membrane potential and intracellular ATP content. Finally, treatment of TrxR2 deficiency cells with N-acetylcysteine (NAC) inhibited mitochondrial ROS production and chondrocyte apoptosis. NAC also prevented chondrogenic differentiation of TrxR2 deficiency cells by suppression of ECM gene expression, GAGs accumulation and mineralization, as well as attenuation of Akt signaling. Thus, TrxR2-mediated mitochondrial integrity is indispensable for chondrogenic differentiation of ATDC5 cells. TrxR2 deficiency-induced impaired proliferation and death of chondrocytes may be the pathological mechanism of the osteoarthropathy due to Se deficiency. Notably, this study also uncover the roles of

  17. Oxygen tension regulates the osteogenic, chondrogenic and endochondral phenotype of bone marrow derived mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Sheehy, Eamon J.; Buckley, Conor T. [Trinity Centre for Bioengineering, School of Engineering, Trinity College Dublin, Dublin 2 (Ireland); Kelly, Daniel J., E-mail: kellyd9@tcd.ie [Trinity Centre for Bioengineering, School of Engineering, Trinity College Dublin, Dublin 2 (Ireland)

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Expansion in low oxygen enhances MSC proliferation and osteogenesis. Black-Right-Pointing-Pointer Differentiation in low oxygen enhances chondrogenesis and suppresses hypertrophy. Black-Right-Pointing-Pointer Oxygen can regulate the MSC phenotype for use in tissue engineering applications. -- Abstract: The local oxygen tension is a key regulator of the fate of mesenchymal stem cells (MSCs). The objective of this study was to investigate the effect of a low oxygen tension during expansion and differentiation on the proliferation kinetics as well as the subsequent osteogenic and chondrogenic potential of MSCs. We first hypothesised that expansion in a low oxygen tension (5% pO{sub 2}) would improve both the subsequent osteogenic and chondrogenic potential of MSCs compared to expansion in a normoxic environment (20% pO{sub 2}). Furthermore, we hypothesised that chondrogenic differentiation in a low oxygen environment would suppress hypertrophy of MSCs cultured in both pellets and hydrogels used in tissue engineering strategies. MSCs expanded at 5% pO{sub 2} proliferated faster forming larger colonies, resulting in higher cell yields. Expansion at 5% pO{sub 2} also enhanced subsequent osteogenesis of MSCs, whereas differentiation at 5% pO{sub 2} was found to be a more potent promoter of chondrogenesis than expansion at 5% pO{sub 2}. Greater collagen accumulation, and more intense staining for collagen types I and X, was observed in pellets maintained at 20% pO{sub 2} compared to 5% pO{sub 2}. Both pellets and hydrogels stained more intensely for type II collagen when undergoing chondrogenesis in a low oxygen environment. Differentiation at 5% pO{sub 2} also appeared to inhibit hypertrophy in both pellets and hydrogels, as demonstrated by reduced collagen type X and Alizarin Red staining and alkaline phosphatase activity. This study demonstrates that the local oxygen environment can be manipulated in vitro to either stabilise a

  18. The assessment of natural scaffolds ability in chondrogenic ...

    African Journals Online (AJOL)

    Arun Kumar Agnihotri

    1Stem Cell Laboratory, The Academic Center for Education, Culture and Research, ... between alginate and agarose groups in maintaining cells viable but, about chondrogenic .... Absorbance at 570 nm was measured on a ... as small cells with little cytoplasm and elliptic ... high density of cells and suitable interaction.

  19. A metabolomics study of the inhibitory effect of 17-beta-estradiol on osteoclast proliferation and differentiation.

    Science.gov (United States)

    Liu, Xiaoyan; Liu, Yanqiu; Cheng, Mengchun; Zhang, Xiaozhe; Xiao, Hongbin

    2015-02-01

    Estradiol is a major drug used clinically to alleviate osteoporosis, partly through inhibition of the activity of osteoclasts, which play a crucial role in bone resorption. So far, little is known about the effects of estradiol on osteoclast metabolism. In this study, ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC/MS)-based metabolomics strategy was used to investigate the metabolite response to 17β-estradiol in mouse osteoclast RAW264.7, a commonly used cell model for studying osteoporosis. Our results showed that the application of estradiol altered the levels of 27 intracellular metabolites, including lysophosphatidylcholines (LysoPCs), other lipids and amino acid derivants. The changes of all the 27 metabolites were observed in the study of estradiol induced osteoclast proliferation inhibition (1 μM estradiol applied), while the changes of only 18 metabolites were observed in the study of differentiation inhibition (0.1 μM estradiol applied). Further pathway impact analysis determined glycerophospholipid metabolism as the main potential target pathway of estradiol, which was further confirmed by LCAT (phosphatidylcholine-sterol acyltransferase) activity changes and lipid peroxidative product (MDA, methane dicarboxylic aldehyde) changes caused by estradiol. Additionally, we found that estradiol significantly decreased intracellular oxidative stress during cell proliferation but not during cell differentiation. Our study suggested that estradiol generated a highly condition-dependent influence on osteoclast metabolism.

  20. HIF-1α as a Regulator of BMP2-Induced Chondrogenic Differentiation, Osteogenic Differentiation, and Endochondral Ossification in Stem Cells

    Directory of Open Access Journals (Sweden)

    Nian Zhou

    2015-04-01

    Full Text Available Background/Aims: Joint cartilage defects are difficult to treat due to the limited self-repair capacities of cartilage. Cartilage tissue engineering based on stem cells and gene enhancement is a potential alternative for cartilage repair. Bone morphogenetic protein 2 (BMP2 has been shown to induce chondrogenic differentiation in mesenchymal stem cells (MSCs; however, maintaining the phenotypes of MSCs during cartilage repair since differentiation occurs along the endochondral ossification pathway. In this study, hypoxia inducible factor, or (HIF-1α, was determined to be a regulator of BMP2-induced chondrogenic differentiation, osteogenic differentiation, and endochondral bone formation. Methods: BMP2 was used to induce chondrogenic and osteogenic differentiation in stem cells and fetal limb development. After HIF-1α was added to the inducing system, any changes in the differentiation markers were assessed. Results: HIF-1α was found to potentiate BMP2-induced Sox9 and the expression of chondrogenesis by downstream markers, and inhibit Runx2 and the expression of osteogenesis by downstream markers in vitro. In subcutaneous stem cell implantation studies, HIF-1α was shown to potentiate BMP2-induced cartilage formation and inhibit endochondral ossification during ectopic bone/cartilage formation. In the fetal limb culture, HIF-1α and BMP2 synergistically promoted the expansion of the proliferating chondrocyte zone and inhibited chondrocyte hypertrophy and endochondral ossification. Conclusion: The results of this study indicated that, when combined with BMP2, HIF-1α induced MSC differentiation could become a new method of maintaining cartilage phenotypes during cartilage tissue engineering.

  1. Purinergic responses of chondrogenic stem cells to dynamic loading

    Directory of Open Access Journals (Sweden)

    Gađanski Ivana

    2013-01-01

    Full Text Available In habitually loaded tissues, dynamic loading can trigger ATP (adenosine 5’- triphosphate release to extracellular environment, and result in calcium signaling via ATP binding to purine P2 receptors1. In the current study we have compared purinergic responses (ATP release of two types of cells: bovine chondrocytes (bCHs and human mesenchymal stem cells (hMSC that were encapsulated in agarose and subjected to dynamic loading. Both cell types were cultured under chondrogenic conditions, and their responses to loading were evaluated by ATP release assay in combination with connexin (Cx-sensitive fluorescent dye (Lucifer Yellow - LY and a Cx-hemichannel blocker (Flufenamic acid - FFA. In response to dynamic loading, chondrogenic hMSCs released significantly higher amounts of ATP (5-fold in comparison to the bCHs early in culture (day 2. Triggering of LY uptake in the bCHs and hMSCs by dynamic loading implies opening of the Cx-hemichannels. However, the number of LY-positive cells in hMSC-constructs was 2.5-fold lower compared to the loaded bCH-constructs, suggesting utilization of additional mechanisms of ATP release. Cx-reactive sites were detected in both bCHs and hMSCs-constructs. FFA application led to reduced ATP release both in bCHs and hMSCs, which confirms the involvement of connexin hemichannels, with more prominent effects in bCHs than in hMSCs, further implying the existence of additional mechanism of ATP release in chondrogenic hMSCs. Taken together, these results indicate stronger purinergic response to dynamic loading of chondrogenic hMSCs than primary chondrocytes, by activation of connexin hemichannels and additional mechanisms of ATP release. [Projekat Ministrastva nauke Republike Srbije, ON174028 i br. III41007

  2. PRC1 Prevents Replication Stress during Chondrogenic Transit Amplification

    Directory of Open Access Journals (Sweden)

    Frank Spaapen

    2017-12-01

    Full Text Available Transit amplification (TA, a state of combined, rapid proliferative expansion and differentiation of stem cell-descendants, remains poorly defined at the molecular level. The Polycomb Repressive Complex 1 (PRC1 protein BMI1 has been localized to TA compartments, yet its exact role in TA is unclear. PRC1 proteins control gene expression, cell proliferation and DNA-damage repair. Coordination of such DNA-templated activities during TA is predicted to be crucial to support DNA replication and differentiation-associated transcriptional programming. We here examined whether chondrogenesis provides a relevant biological context for synchronized coordination of these chromatin-based tasks by BMI1. Taking advantage of a prominently featuring TA-phase during chondrogenesis in vitro and in vivo, we here report that TA is completely dependent on intact PRC1 function. BMI1-depleted chondrogenic progenitors rapidly accumulate double strand DNA breaks during DNA replication, present massive non-H3K27me3-directed transcriptional deregulation and fail to undergo chondrogenic TA. Genome-wide accumulation of Topoisomerase 2α and Geminin suggests a model in which PRC1 synchronizes replication and transcription during rapid chondrogenic progenitor expansion. Our combined data reveals for the first time a vital cell-autonomous role for PRC1 during chondrogenesis. We provide evidence that chondrocyte hyper-replication and hypertrophy represent a unique example of programmed senescence in vivo. These findings provide new perspectives on PRC1 function in development and disease.

  3. Chondrogenic potential of canine articular cartilage derived cells (cACCs

    Directory of Open Access Journals (Sweden)

    Nowak Urszula

    2016-01-01

    Full Text Available In the present paper, the potential of canine articular cartilage-derived cells (cACCs for chondrogenic differentiation was evaluated. The effectiveness of cACCs’ lineage commitment was analyzed after 14 days of culture in chondorgenic and non-chondrogenic conditions. Formation of proteoglycan-rich extracellular matrix was assessed using histochemical staining – Alcian Blue and Safranin-O, while elemental composition was determined by means of SEM-EDX. Additionally, ultrastructure of cACCs was evaluated using TEM. The expression of genes involved in chondrogenesis was monitored with quantitative Real Time PCR. Results obtained indicate that the potential of cACCs for cartilagous extracellular matrix formation may be maintained only in chondrogenic cultures. The formation of specific chondro-nodules was not observed in a non-chondrogenic culture environment. The analysis of cACCs’ ultrastructure, both in non-chondrogenic and chondrogenic cultures, revealed well-developed rough endoplasmatic reticulum and presence of mitochondria. The cACCs in chondrogenic medium shed an increased number of microvesicles. Furthermore, it was shown that the extracellular matrix of cACCs in chondrogenic cultures is rich in potassium and molybdenum. Additionally, it was determined that gene expression of collagen type II, aggrecan and SOX-9 was significantly increased during chondrogenic differentiation of cACCs. Results obtained indicate that the culture environment may significantly influence the cartilage phenotype of cACCs during long term culture.

  4. Mesenchymal stem cells maintain TGF-beta-mediated chondrogenic phenotype in alginate bead culture

    DEFF Research Database (Denmark)

    Mehlhorn, A T; Schmal, H; Kaiser, S

    2006-01-01

    cultured in osteogenic medium after TGF-beta-mediated chondroinduction. Gene expression of col2a1, aggrecan, COMP, alkaline phosphatase (AP), and correlating protein synthesis was analyzed. After short-term stimulation with TGF-beta, MSCs maintained a chondrogenic phenotype. Chondrogenic gene expression...

  5. Effects of estradiol on radiation-induced apoptosis in immunocytes of mouse

    International Nuclear Information System (INIS)

    Wu Wei; Yang Rujun; Kong Xiantao; Zhang Lingzhen; Li Bolong; Cai Jianming

    2000-01-01

    Objective: To assess the effects of estradiol on 60 Co γ-radiation induced apoptosis of splenic lymphocytes and thymocytes, and surface molecule expression of splenic lymphocytes. Methods: Mice were whole body irradiated with 4.0 Gy γ-rays. By flow cytometry and electrophoretic analysis of DNA, the changes in apoptosis of mouse immunocytes were determined. The splenic lymphocytes were analyzed by flow cytometry with fluorescent monoclonal antibodies. Results: 10 days after administration of estradiol, the characteristic DNA ladder in mice 8h after irradiation was minor than in mice without estradiol administration,indicating that the apoptotic rate reduced on flow cytometry. CD4+ T cells, CD8+ T cells and IgM+ B cells up regulated Fas, CD25 and CD69 expression, but did not so in the estradiol treated mice. Conclusion: Estradiol can block CD25, CD69 and Fas overexpression, thereby inhibiting Fas mediated apoptosis induced by γ-irradiation

  6. Transfer of estradiol to human milk. [Radioimmunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Nilsson, S.; Nygren, K.G.; Johansson, E.D.B.

    1978-11-15

    A radioimmunoassay for the measurement of estradiol in human milk is evaluated. The detection limit was found to be 25 pg of estradiol per milliliter of milk. In milk samples collected from four lactating women during three to four months and from one pregnant and lactating woman, the concentration of estradiol was found to be below the detection limit of the assay. When six lactating women were given vaginal suppositories containing 50 or 100 mg of estradiol, it was possible to estimate the estradiol concentration in milk. A ratio of transfer of estradiol from plasma to milk during physiologic conditions is calculated to be less than 100 : 10.

  7. Transfer of estradiol to human milk

    International Nuclear Information System (INIS)

    Nilsson, S.; Nygren, K.G.; Johansson, E.D.B.

    1978-01-01

    A radioimmunoassay for the measurement of estradiol in human milk is evaluated. The detection limit was found to be 25 pg of estradiol per milliliter of milk. In milk samples collected from four lactating women during three to four months and from one pregnant and lactating woman, the concentration of estradiol was found to be below the detection limit of the assay. When six lactating women were given vaginal suppositories containing 50 or 100 mg of estradiol, it was possible to estimate the estradiol concentration in milk. A ratio of transfer of estradiol from plasma to milk during physiologic conditions is calculated to be less than 100 : 10

  8. Hormone-dependent nuclear export of estradiol receptor and DNA synthesis in breast cancer cells

    Science.gov (United States)

    Lombardi, Maria; Castoria, Gabriella; Migliaccio, Antimo; Barone, Maria Vittoria; Di Stasio, Rosina; Ciociola, Alessandra; Bottero, Daniela; Yamaguchi, Hiroshi; Appella, Ettore; Auricchio, Ferdinando

    2008-01-01

    In breast cancer cells, cytoplasmic localization of the estradiol receptor α (ERα) regulates estradiol-dependent S phase entry. We identified a nuclear export sequence (NES) in ERα and show that its export is dependent on both estradiol-mediated phosphatidylinositol-3-kinase (PI3K)/AKT activation and chromosome region maintenance 1 (CRM1). A Tat peptide containing the ERα NES disrupts ERα–CRM1 interaction and prevents nuclear export of ERα- and estradiol-induced DNA synthesis. NES-ERα mutants do not exit the nucleus and inhibit estradiol-induced S phase entry; ERα-dependent transcription is normal. ERα is associated with Forkhead proteins in the nucleus, and estradiol stimulates nuclear exit of both proteins. ERα knockdown or ERα NES mutations prevent ERα and Forkhead nuclear export. A mutant of forkhead in rhabdomyosarcoma (FKHR), which cannot be phosphorylated by estradiol-activated AKT, does not associate with ERα and is trapped in the nucleus, blocking S phase entry. In conclusion, estradiol-induced AKT-dependent phosphorylation of FKHR drives its association with ERα, thereby triggering complex export from the nucleus necessary for initiation of DNA synthesis and S phase entry. PMID:18644889

  9. Evidence that 17alpha-estradiol is biologically active in the uterine tissue: Antiuterotonic and antiuterotrophic action

    Directory of Open Access Journals (Sweden)

    Navarrete Erika

    2005-07-01

    Full Text Available Abstract Background 17alpha-Estradiol has been considered as the hormonally inactive isomer of 17beta-estradiol. Recently, nongenomic (smooth muscle relaxation and genomic (light estrogenic activity effects of 17alpha-estradiol have been reported, but no reports have yet determined its possible antiestrogenic activity. Therefore, this study investigated: the nongenomic action of 17alpha-estradiol on uterine contractile activity and its potential agonist-antagonist activity on uterine growth. Methods Uterine rings from rats were isometrically recorded. Different concentrations (0.2–200 microM of 17alpha-estradiol were tested on spontaneous contraction and equimolarly compared with 17beta-estradiol. To examine the mechanism of 17alpha-estradiol action, its effect was studied in presence of beta2-antagonist (propranolol, antiestrogens (tamoxifen and ICI 182,780 or inhibitors of protein synthesis (cycloheximide and transcription (actinomycin D. Moreover, contractions induced by high potassium (KCl solution or calcium in depolarized tissues by KCl-calcium free solution were exposed to 17alpha-estradiol. Collaterally, we performed an uterotrophic assay in adult ovariectomized rats measuring the uterine wet weight. The administration for three days of 0.3 microM/day/Kg 17beta-estradiol was equimolarly compared with the response produced by 17alpha-estradiol. Antiuterotrophic activity was assayed by administration of 0.3 microM/day/Kg 17beta-estradiol and various doses ratios (1:1, 1:3, 1:5, and 1:100 of 17alpha-estradiol. Results The estradiol isomers elicited an immediate relaxation, concentration-dependent and reversible on spontaneous contraction. 17alpha-Estradiol presented lower potency than 17beta-estradiol although it did not antagonize 17beta-estradiol-induced relaxation. Relaxation to 17alpha-estradiol was not inhibited by propranolol, tamoxifen, ICI 182,780, cycloheximide or actinomycin D. The KCl contractions were also sensitive to 17alpha-estradiol

  10. Cartilage Protective and Chondrogenic Capacity of WIN-34B, a New Herbal Agent, in the Collagenase-Induced Osteoarthritis Rabbit Model and in Progenitor Cells from Subchondral Bone

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    Jeong-Eun Huh

    2013-01-01

    Full Text Available We sought to determine the cartilage repair capacity of WIN-34B in the collagenase-induced osteoarthritis rabbit model and in progenitor cells from subchondral bone. The cartilage protective effect of WIN-34B was measured by clinical and histological scores, cartilage area, and proteoglycan and collagen contents in the collagenase-induced osteoarthritis rabbit model. The efficacy of chondrogenic differentiation of WIN-34B was assessed by expression of CD105, CD73, type II collagen, and aggrecan in vivo and was analyzed by the surface markers of progenitor cells, the mRNA levels of chondrogenic marker genes, and the level of proteoglycan, GAG, and type II collagen in vitro. Oral administration of WIN-34B significantly increased cartilage area, and this was associated with the recovery of proteoglycan and collagen content. Moreover, WIN-34B at 200 mg/kg significantly increased the expression of CD105, CD73, type II collagen, and aggrecan compared to the vehicle group. WIN-34B markedly enhanced the chondrogenic differentiation of CD105 and type II collagen in the progenitor cells from subchondral bone. Also, we confirmed that treatment with WIN-34B strongly increased the number of SH-2(CD105 cells and expression type II collagen in subchondral progenitor cells. Moreover, WIN-34B significantly increased proteoglycan, as measured by alcian blue staining; the mRNA level of type II α1 collagen, cartilage link protein, and aggrecan; and the inhibition of cartilage matrix molecules, such as GAG and type II collagen, in IL-1β-treated progenitor cells. These findings suggest that WIN-34B could be a potential candidate for effective anti-osteoarthritic therapy with cartilage repair as well as cartilage protection via enhancement of chondrogenic differentiation in the collagenase-induced osteoarthritis rabbit model and progenitor cells from subchondral bone.

  11. Enhancement of Matrix Metalloproteinase-2 (MMP-2 as a Potential Chondrogenic Marker during Chondrogenic Differentiation of Human Adipose-Derived Stem Cells

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    Yoshie Arai

    2016-06-01

    Full Text Available Human adipose-derived stem cells (hASCs have a capacity to undergo adipogenic, chondrogenic, and osteogenic differentiation. Recently, hASCs were applied to various fields including cell therapy for tissue regeneration. However, it is hard to predict the direction of differentiation of hASCs in real-time. Matrix metalloproteinases (MMPs are one family of proteolytic enzymes that plays a pivotal role in regulating the biology of stem cells. MMPs secreted by hASCs are expected to show different expression patterns depending on the differentiation state of hASCs because biological functions exhibit different patterns during the differentiation of stem cells. Here, we investigated proteolytic enzyme activity, especially MMP-2 activity, in hASCs during their differentiation. The activities of proteolytic enzymes and MMP-2 were higher during chondrogenic differentiation than during adipogenic and osteogenic differentiation. During chondrogenic differentiation, mRNA expression of MMP-2 and the level of the active form of MMP-2 were increased, which also correlated with Col II. It is concluded that proteolytic enzyme activity and the level of the active form of MMP-2 were increased during chondrogenic differentiation, which was accelerated in the presence of Col II protein. According to our findings, MMP-2 could be a candidate maker for real-time detection of chondrogenic differentiation of hASCs.

  12. The Chondrogenic Induction Potential for Bone Marrow-Derived Stem Cells between Autologous Platelet-Rich Plasma and Common Chondrogenic Induction Agents: A Preliminary Comparative Study

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    Shan-zheng Wang

    2015-01-01

    Full Text Available The interests in platelet-rich plasma (PRP and their application in stem cell therapy have contributed to a better understanding of the basic biology of the prochondrogenesis effect on bone marrow-derived stem cells (BMSCs. We aimed at comparing the effect of autologous PRP with common chondrogenic induction agents (CCIAs on the chondrogenic differentiation of BMSCs. Rabbit BMSCs were isolated and characterized by flow cytometry and differentiated towards adipocytes and osteoblasts. The chondrogenic response of BMSCs to autologous PRP and CCIAs which included transforming growth factor-β1 (TGF-β1, dexamethasone (DEX, and vitamin C (Vc was examined by cell pellet culture. The isolated BMSCs after two passages highly expressed CD29 and CD44 but minimally expressed CD45. The osteogenic and adipogenic differentiation potentials of the isolated BMSCs were also confirmed. Compared with common CCIAs, autologous PRP significantly upregulated the chondrogenic related gene expression, including Col-2, AGC, and Sox-9. Osteogenic related gene expression, including Col-1 and OCN, was not of statistical significance between these two groups. Thus, our data shows that, compared with common chondrogenic induction agents, autologous PRP can be more effective in promoting the chondrogenesis of BMSCs.

  13. Synthesis of 123I-16 iodo estradiol

    International Nuclear Information System (INIS)

    Therain, F.; Gros, J.; Souchu, A.

    1982-01-01

    16α iodo estradiol has been demonstrated to have as good an affinity as estradiol for estrogen-receptors and, labeled with iodine 123, may provide a good scanning agent fot visualisation of tissues containing estrogen-repectors, especially mammary tumors. 123 I-16α iodo estradiol has been synthesized by an halogen exchange of 16ν bromo estradiol according to the procedure described by Hochberg for 125 I-16α iodo estradiol labeling. Radiochemical yields are much lower than with iodine 125 (1 to 30%) and extremely variable. Specific activity range from 1,000 to 2,000 Ci/mmole [fr

  14. Direct induction of chondrogenic cells from human dermal fibroblast culture by defined factors.

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    Hidetatsu Outani

    Full Text Available The repair of large cartilage defects with hyaline cartilage continues to be a challenging clinical issue. We recently reported that the forced expression of two reprogramming factors (c-Myc and Klf4 and one chondrogenic factor (SOX9 can induce chondrogenic cells from mouse dermal fibroblast culture without going through a pluripotent state. We here generated induced chondrogenic (iChon cells from human dermal fibroblast (HDF culture with the same factors. We developed a chondrocyte-specific COL11A2 promoter/enhancer lentiviral reporter vector to select iChon cells. The human iChon cells expressed marker genes for chondrocytes but not fibroblasts, and were derived from non-chondrogenic COL11A2-negative cells. The human iChon cells formed cartilage but not tumors in nude mice. This approach could lead to the preparation of cartilage directly from skin in human, without going through pluripotent stem cells.

  15. Cartilage regeneration by chondrogenic induced adult stem cells in osteoarthritic sheep model.

    Science.gov (United States)

    Ude, Chinedu C; Sulaiman, Shamsul B; Min-Hwei, Ng; Hui-Cheng, Chen; Ahmad, Johan; Yahaya, Norhamdan M; Saim, Aminuddin B; Idrus, Ruszymah B H

    2014-01-01

    In this study, Adipose stem cells (ADSC) and bone marrow stem cells (BMSC), multipotent adult cells with the potentials for cartilage regenerations were induced to chondrogenic lineage and used for cartilage regenerations in surgically induced osteoarthritis in sheep model. Osteoarthritis was induced at the right knee of sheep by complete resection of the anterior cruciate ligament and medial meniscus following a 3-weeks exercise regimen. Stem cells from experimental sheep were culture expanded and induced to chondrogenic lineage. Test sheep received a single dose of 2 × 10(7) autologous PKH26-labelled, chondrogenically induced ADSCs or BMSCs as 5 mls injection, while controls received 5 mls culture medium. The proliferation rate of ADSCs 34.4 ± 1.6 hr was significantly higher than that of the BMSCs 48.8 ± 5.3 hr (P = 0.008). Chondrogenic induced BMSCs had significantly higher expressions of chondrogenic specific genes (Collagen II, SOX9 and Aggrecan) compared to chondrogenic ADSCs (P = 0.031, 0.010 and 0.013). Grossly, the treated knee joints showed regenerated de novo cartilages within 6 weeks post-treatment. On the International Cartilage Repair Society grade scores, chondrogenically induced ADSCs and BMSCs groups had significantly lower scores than controls (P = 0.0001 and 0.0001). Fluorescence of the tracking dye (PKH26) in the injected cells showed that they had populated the damaged area of cartilage. Histological staining revealed loosely packed matrixes of de novo cartilages and immunostaining demonstrated the presence of cartilage specific proteins, Collagen II and SOX9. Autologous chondrogenically induced ADSCs and BMSCs could be promising cell sources for cartilage regeneration in osteoarthritis.

  16. Cartilage regeneration by chondrogenic induced adult stem cells in osteoarthritic sheep model.

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    Chinedu C Ude

    Full Text Available OBJECTIVES: In this study, Adipose stem cells (ADSC and bone marrow stem cells (BMSC, multipotent adult cells with the potentials for cartilage regenerations were induced to chondrogenic lineage and used for cartilage regenerations in surgically induced osteoarthritis in sheep model. METHODS: Osteoarthritis was induced at the right knee of sheep by complete resection of the anterior cruciate ligament and medial meniscus following a 3-weeks exercise regimen. Stem cells from experimental sheep were culture expanded and induced to chondrogenic lineage. Test sheep received a single dose of 2 × 10(7 autologous PKH26-labelled, chondrogenically induced ADSCs or BMSCs as 5 mls injection, while controls received 5 mls culture medium. RESULTS: The proliferation rate of ADSCs 34.4 ± 1.6 hr was significantly higher than that of the BMSCs 48.8 ± 5.3 hr (P = 0.008. Chondrogenic induced BMSCs had significantly higher expressions of chondrogenic specific genes (Collagen II, SOX9 and Aggrecan compared to chondrogenic ADSCs (P = 0.031, 0.010 and 0.013. Grossly, the treated knee joints showed regenerated de novo cartilages within 6 weeks post-treatment. On the International Cartilage Repair Society grade scores, chondrogenically induced ADSCs and BMSCs groups had significantly lower scores than controls (P = 0.0001 and 0.0001. Fluorescence of the tracking dye (PKH26 in the injected cells showed that they had populated the damaged area of cartilage. Histological staining revealed loosely packed matrixes of de novo cartilages and immunostaining demonstrated the presence of cartilage specific proteins, Collagen II and SOX9. CONCLUSION: Autologous chondrogenically induced ADSCs and BMSCs could be promising cell sources for cartilage regeneration in osteoarthritis.

  17. Ultra-structural changes and expression of chondrogenic and hypertrophic genes during chondrogenic differentiation of mesenchymal stromal cells in alginate beads

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    Havva Dashtdar

    2016-03-01

    Full Text Available Chondrogenic differentiation of mesenchymal stromal cells (MSCs in the form of pellet culture and encapsulation in alginate beads has been widely used as conventional model for in vitro chondrogenesis. However, comparative characterization between differentiation, hypertrophic markers, cell adhesion molecule and ultrastructural changes during alginate and pellet culture has not been described. Hence, the present study was conducted comparing MSCs cultured in pellet and alginate beads with monolayer culture. qPCR was performed to assess the expression of chondrogenic, hypertrophic, and cell adhesion molecule genes, whereas transmission electron microscopy (TEM was used to assess the ultrastructural changes. In addition, immunocytochemistry for Collagen type II and aggrecan and glycosaminoglycan (GAG analysis were performed. Our results indicate that pellet and alginate bead cultures were necessary for chondrogenic differentiation of MSC. It also indicates that cultures using alginate bead demonstrated significantly higher (p < 0.05 chondrogenic but lower hypertrophic (p < 0.05 gene expressions as compared with pellet cultures. N-cadherin and N-CAM1 expression were up-regulated in second and third weeks of culture and were comparable between the alginate bead and pellet culture groups, respectively. TEM images demonstrated ultrastructural changes resembling cell death in pellet cultures. Our results indicate that using alginate beads, MSCs express higher chondrogenic but lower hypertrophic gene expression. Enhanced production of extracellular matrix and cell adhesion molecules was also observed in this group. These findings suggest that alginate bead culture may serve as a superior chondrogenic model, whereas pellet culture is more appropriate as a hypertrophic model of chondrogenesis.

  18. Enamel Matrix Derivative has No Effect on the Chondrogenic Differentiation of Mesenchymal Stem Cells

    Energy Technology Data Exchange (ETDEWEB)

    Groeneveldt, Lisanne C.; Knuth, Callie; Witte-Bouma, Janneke [Department of Oral and Maxillofacial Surgery, Special Dental Care and Orthodontics, Erasmus University Medical Center, Rotterdam (Netherlands); O’Brien, Fergal J. [Tissue Engineering Research Group, Department of Anatomy, Royal College of Surgeons in Ireland, Dublin (Ireland); Wolvius, Eppo B.; Farrell, Eric, E-mail: e.farrell@erasmusmc.nl [Department of Oral and Maxillofacial Surgery, Special Dental Care and Orthodontics, Erasmus University Medical Center, Rotterdam (Netherlands)

    2014-09-02

    Background: Treatment of large bone defects due to trauma, tumor resection, or congenital abnormalities is challenging. Bone tissue engineering using mesenchymal stem cells (MSCs) represents a promising treatment option. However, the quantity and quality of engineered bone tissue are not sufficient to fill large bone defects. The aim of this study was to determine if the addition of enamel matrix derivative (EMD) improves in vitro chondrogenic priming of MSCs to ultimately improve in vivo MSC mediated endochondral bone formation. Methods: MSCs were chondrogenically differentiated in 2.0 × 10{sup 5} cell pellets in medium supplemented with TGFβ3 in the absence or presence of 1, 10, or 100 μg/mL EMD. Samples were analyzed for gene expression of RUNX2, Col II, Col X, and Sox9. Protein and glycoaminoglycan (GAG) production were also investigated via DMB assays, histology, and immunohistochemistry. Osteogenic and adipogenic differentiation capacity were also assessed. Results: The addition of EMD did not negatively affect chondrogenic differentiation of adult human MSCs. EMD did not appear to alter GAG production or expression of chondrogenic genes. Osteogenic and adipogenic differentiation were also unaffected though a trend toward decreased adipogenic gene expression was observed. Conclusion: EMD does not affect chondrogenic differentiation of adult human MSCs. As such the use of EMD in combination with chondrogenically primed MSCs for periodontal bone tissue repair is unlikely to have negative effects on MSC differentiation.

  19. Enamel Matrix Derivative has No Effect on the Chondrogenic Differentiation of Mesenchymal Stem Cells

    International Nuclear Information System (INIS)

    Groeneveldt, Lisanne C.; Knuth, Callie; Witte-Bouma, Janneke; O’Brien, Fergal J.; Wolvius, Eppo B.; Farrell, Eric

    2014-01-01

    Background: Treatment of large bone defects due to trauma, tumor resection, or congenital abnormalities is challenging. Bone tissue engineering using mesenchymal stem cells (MSCs) represents a promising treatment option. However, the quantity and quality of engineered bone tissue are not sufficient to fill large bone defects. The aim of this study was to determine if the addition of enamel matrix derivative (EMD) improves in vitro chondrogenic priming of MSCs to ultimately improve in vivo MSC mediated endochondral bone formation. Methods: MSCs were chondrogenically differentiated in 2.0 × 10 5 cell pellets in medium supplemented with TGFβ3 in the absence or presence of 1, 10, or 100 μg/mL EMD. Samples were analyzed for gene expression of RUNX2, Col II, Col X, and Sox9. Protein and glycoaminoglycan (GAG) production were also investigated via DMB assays, histology, and immunohistochemistry. Osteogenic and adipogenic differentiation capacity were also assessed. Results: The addition of EMD did not negatively affect chondrogenic differentiation of adult human MSCs. EMD did not appear to alter GAG production or expression of chondrogenic genes. Osteogenic and adipogenic differentiation were also unaffected though a trend toward decreased adipogenic gene expression was observed. Conclusion: EMD does not affect chondrogenic differentiation of adult human MSCs. As such the use of EMD in combination with chondrogenically primed MSCs for periodontal bone tissue repair is unlikely to have negative effects on MSC differentiation.

  20. Mechanism of Estradiol-Induced Block of Voltage-Gated K+ Currents in Rat Medial Preoptic Neurons

    Science.gov (United States)

    Druzin, Michael; Malinina, Evgenya; Grimsholm, Ola; Johansson, Staffan

    2011-01-01

    The present study was conducted to characterize possible rapid effects of 17-β-estradiol on voltage-gated K+ channels in preoptic neurons and, in particular, to identify the mechanisms by which 17-β-estradiol affects the K+ channels. Whole-cell currents from dissociated rat preoptic neurons were studied by perforated-patch recording. 17-β-estradiol rapidly (within seconds) and reversibly reduced the K+ currents, showing an EC50 value of 9.7 µM. The effect was slightly voltage dependent, but independent of external Ca2+, and not sensitive to an estrogen-receptor blocker. Although 17-α-estradiol also significantly reduced the K+ currents, membrane-impermeant forms of estradiol did not reduce the K+ currents and other estrogens, testosterone and cholesterol were considerably less effective. The reduction induced by estradiol was overlapping with that of the KV-2-channel blocker r-stromatoxin-1. The time course of K+ current in 17-β-estradiol, with a time-dependent inhibition and a slight dependence on external K+, suggested an open-channel block mechanism. The properties of block were predicted from a computational model where 17-β-estradiol binds to open K+ channels. It was concluded that 17-β-estradiol rapidly reduces voltage-gated K+ currents in a way consistent with an open-channel block mechanism. This suggests a new mechanism for steroid action on ion channels. PMID:21625454

  1. Cartilage tumors. Pathology and radiomorphology; Chondrogene Knochentumoren. Pathologie und Radiomorphologie

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    Uhl, M. [RKK-Klinikum Freiburg, Klinik fuer Diagnostische und Interventionelle Radiologie, Kinderradiologie und Neuroradiologie SJK, Freiburg (Germany); Herget, G. [Universitaetsklinik Freiburg, Department Orthopaedie und Traumatologie, Freiburg (Germany); Kurz, P. [Universitaetsklinik Freiburg, Pathologisches Institut, Freiburg (Germany)

    2016-06-15

    Primary cartilage-forming tumors of the bone are frequent entities in the daily work of skeletal radiologists. This article describes the correlation of pathology and radiology in cartilage-forming skeletal tumors, in particular, enchondroma, osteochondroma, periosteal chondromas, chondroblastoma and various forms of chondrosarcoma. After reading, the radiologist should be able to deduce the different patterns of cartilage tumors on radiographs, CT, and MRI from the pathological aspects. Differentiation of enchondroma and chondrosarcoma is a frequent diagnostic challenge. Some imaging parameters, e. g., deep cortical scalloping (more than two thirds of the cortical thickness), cortical destruction, or a soft-tissue mass, are features of a sarcoma. Osteochondromas are bony protrusions with a continuous extension of bone marrow from the parent bone, the host cortical bone runs continuously from the osseous surface of the tumor into the shaft of the osteochondroma and the osteochondroma has a cartilage cap. Chondromyxoid fibromas are well-defined lytic and eccentric lesions of the metaphysis of the long bones, with nonspecific MRI findings. Chondroblastomas have a strong predilection for the epiphysis of long tubular bones and develop an intense perifocal bone marrow edema. Dedifferentiated chondrosarcomas are bimorphic lesions with a low-grade chondrogenic component and a high-grade noncartilaginous component. Most chondrogenic tumors have a predilection with regard to site and age at manifestation. (orig.) [German] Primaere knorpelbildende Tumoren sind haeufige Entitaeten in der taeglichen Arbeit des Radiologen. Der Beitrag beschreibt die Korrelation von Pathologie und Radiologie knorpelbildender Skeletttumoren, insbesondere von Enchondrom, Osteochondrom, periostalem Chondrom, Chondroblastom, und verschiedenen Varianten des Chondrosarkoms. Nach Lesen des Beitrags kann der Radiologe die verschiedenen typischen Muster knorpelbildender Tumoren im Roentgenbild

  2. Scaffold composition affects cytoskeleton organization, cell-matrix interaction and the cellular fate of human mesenchymal stem cells upon chondrogenic differentiation.

    Science.gov (United States)

    Li, Yuk Yin; Choy, Tze Hang; Ho, Fu Chak; Chan, Pui Barbara

    2015-06-01

    The stem cell niche, or microenvironment, consists of soluble, matrix, cell and mechanical factors that together determine the cellular fates and/or differentiation patterns of stem cells. Collagen and glycosaminoglycans (GAGs) are important scaffolding materials that can mimic the natural matrix niche. Here, we hypothesize that imposing changes in the scaffold composition or, more specifically, incorporating GAGs into the collagen meshwork, will affect the morphology, cytoskeletal organization and integrin expression profiles, and hence the fate of human mesenchymal stem cells (MSCs) upon the induction of differentiation. Using chondrogenesis as an example, we microencapsulated MSCs in three scaffold systems that had varying matrix compositions: collagen alone (C), aminated collagen (AC) and aminated collagen with GAGs (ACG). We then induced the MSCs to differentiate toward a chondrogenic lineage, after which, we characterized the cell viability and morphology, as well as the level of cytoskeletal organization and the integrin expression profile. We also studied the fate of the MSCs by evaluating the major chondrogenic markers at both the gene and protein level. In C, MSC chondrogenesis was successfully induced and MSCs that spread in the scaffolds had a clear actin cytoskeleton; they expressed integrin α2β1, α5 and αv; promoted sox9 nuclear localization transcription activation; and upregulated the expression of chondrogenic matrix markers. In AC, MSC chondrogenesis was completely inhibited but the scaffold still supported cell survival. The MSCs did not spread and they had no actin cytoskeleton; did not express integrin α2 or αv; they failed to differentiate into chondrogenic lineage cells even on chemical induction; and there was little colocalization or functional interaction between integrin α5 and fibronectin. In ACG, although the MSCs did not express integrin α2, they did express integrin αv and there was strong co-localization and hence functional

  3. The Effects of the WNT-Signaling Modulators BIO and PKF118-310 on the Chondrogenic Differentiation of Human Mesenchymal Stem Cells

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    Xiaobin Huang

    2018-02-01

    Full Text Available Mesenchymal stem cells (MSCs are multipotent cells, mainly from bone marrow, and an ideal source of cells in bone and cartilage tissue engineering. A study of the chondrogenic differentiation of MSCs is of particular interest for MSCs-based cartilage regeneration. In this study, we aimed to optimize the conditions for the chrondogenic differentiation of MSCs by regulating WNT signaling using the small molecule WNT inhibitor PKF118-310 and activator BIO. Human mesenchymal stem cells (hMSCs were isolated from bone marrow aspirates and cultured in hMSCs proliferation medium. Pellet culture was subsequently established for three-dimensional chondrogenic differentiation of 5 weeks. WNT signaling was increased by the small molecule glycogen synthase kinase-3 inhibitor 6-bromoindirubin-3-oxim (BIO and decreased by the WNT inhibitor PKF118-310 (PKF. The effects of BIO and PKF on the chondrogenesis of hMSCs was examined by real-time PCR, histological methods, and ELISA. We found that activation of canonical WNT-signaling by BIO significantly downregulated the expression of cartilage-specific genes SOX9, COL2A1, and ACAN, and matrix metalloproteinase genes MMP1/3/9/13, but increased ADAMTS 4/5. Inhibition of WNT signaling by PKF increased the expression of SOX9, COL2A1, ACAN, and MMP9, but decreased MMP13 and ADAMTS4/5. In addition, a high level of WNT signaling induced the expression of hypertrophic markers COL10A1, ALPL, and RUNX2, the dedifferentiation marker COL1A1, and glycolysis genes GULT1 and PGK1. Deposition of glycosaminoglycan (GAG and collagen type II in the pellet matrix was significantly lost in the BIO-treated group and increased in the PKF-treated group. The protein level of COL10A1 was also highly induced in the BIO group. Interestingly, BIO decreased the number of apoptotic cells while PKF significantly induced apoptosis during chondrogenesis. The natural WNT antagonist DKK1 and the protein level of MMP1 in the pellet culture medium were

  4. The Effects of the WNT-Signaling Modulators BIO and PKF118-310 on the Chondrogenic Differentiation of Human Mesenchymal Stem Cells.

    Science.gov (United States)

    Huang, Xiaobin; Zhong, Leilei; Hendriks, Jan; Post, Janine N; Karperien, Marcel

    2018-02-13

    Mesenchymal stem cells (MSCs) are multipotent cells, mainly from bone marrow, and an ideal source of cells in bone and cartilage tissue engineering. A study of the chondrogenic differentiation of MSCs is of particular interest for MSCs-based cartilage regeneration. In this study, we aimed to optimize the conditions for the chrondogenic differentiation of MSCs by regulating WNT signaling using the small molecule WNT inhibitor PKF118-310 and activator BIO. Human mesenchymal stem cells (hMSCs) were isolated from bone marrow aspirates and cultured in hMSCs proliferation medium. Pellet culture was subsequently established for three-dimensional chondrogenic differentiation of 5 weeks. WNT signaling was increased by the small molecule glycogen synthase kinase-3 inhibitor 6-bromoindirubin-3-oxim (BIO) and decreased by the WNT inhibitor PKF118-310 (PKF). The effects of BIO and PKF on the chondrogenesis of hMSCs was examined by real-time PCR, histological methods, and ELISA. We found that activation of canonical WNT-signaling by BIO significantly downregulated the expression of cartilage-specific genes SOX9 , COL2A1, and ACAN , and matrix metalloproteinase genes MMP1/3/9/13, but increased ADAMTS 4/5 . Inhibition of WNT signaling by PKF increased the expression of SOX9 , COL2A1 , ACAN , and MMP9, but decreased MMP13 and ADAMTS4/5 . In addition, a high level of WNT signaling induced the expression of hypertrophic markers COL10A1, ALPL , and RUNX2, the dedifferentiation marker COL1A1 , and glycolysis genes GULT1 and PGK1 . Deposition of glycosaminoglycan (GAG) and collagen type II in the pellet matrix was significantly lost in the BIO-treated group and increased in the PKF-treated group. The protein level of COL10A1 was also highly induced in the BIO group. Interestingly, BIO decreased the number of apoptotic cells while PKF significantly induced apoptosis during chondrogenesis. The natural WNT antagonist DKK1 and the protein level of MMP1 in the pellet culture medium were

  5. Characterization of human adipose-derived stem cells and expression of chondrogenic genes during induction of cartilage differentiation.

    Science.gov (United States)

    Hamid, Adila A; Idrus, Ruszymah Bt Hj; Saim, Aminuddin Bin; Sathappan, Somasumdaram; Chua, Kien-Hui

    2012-01-01

    Understanding the changes in chondrogenic gene expression that are involved in the differentiation of human adipose-derived stem cells to chondrogenic cells is important prior to using this approach for cartilage repair. The aims of the study were to characterize human adipose-derived stem cells and to examine chondrogenic gene expression after one, two, and three weeks of induction. Human adipose-derived stem cells at passage 4 were evaluated by flow cytometry to examine the expression of surface markers. These adipose-derived stem cells were tested for adipogenic and osteogenic differentiation capacity. Ribonucleic acid was extracted from the cells for quantitative polymerase chain reaction analysis to determine the expression levels of chondrogenic genes after chondrogenic induction. Human adipose-derived stem cells were strongly positive for the mesenchymal markers CD90, CD73, CD44, CD9, and histocompatibility antigen and successfully differentiated into adipogenic and osteogenic lineages. The human adipose-derived stem cells aggregated and formed a dense matrix after chondrogenic induction. The expression of chondrogenic genes (collagen type II, aggrecan core protein, collagen type XI, COMP, and ELASTIN) was significantly higher after the first week of induction. However, a significantly elevated expression of collagen type X was observed after three weeks of chondrogenic induction. Human adipose-derived stem cells retain stem cell characteristics after expansion in culture to passage 4 and serve as a feasible source of cells for cartilage regeneration. Chondrogenesis in human adipose-derived stem cells was most prominent after one week of chondrogenic induction.

  6. Curcumin mediated suppression of nuclear factor-κB promotes chondrogenic differentiation of mesenchymal stem cells in a high-density co-culture microenvironment.

    Science.gov (United States)

    Buhrmann, Constanze; Mobasheri, Ali; Matis, Ulrike; Shakibaei, Mehdi

    2010-01-01

    Osteoarthritis (OA) and rheumatoid arthritis (RA) are characterised by joint inflammation and cartilage degradation. Although mesenchymal stem cell (MSC)-like progenitors are resident in the superficial zone of articular cartilage, damaged tissue does not possess the capacity for regeneration. The high levels of pro-inflammatory cytokines present in OA/RA joints may impede the chondrogenic differentiation of these progenitors. Interleukin (IL)-1β activates the transcription factor nuclear factor-κB (NF-κB), which in turn activates proteins involved in matrix degradation, inflammation and apoptosis. Curcumin is a phytochemical capable of inhibiting IL-1β-induced activation of NF-κB and expression of apoptotic and pro-inflammatory genes in chondrocytes. Therefore, the aim of the present study was to evaluate the influence of curcumin on IL-1β-induced NF-κB signalling pathway in MSCs during chondrogenic differentiation. MSCs were either cultured in a ratio of 1:1 with primary chondrocytes in high-density culture or cultured alone in monolayer with/without curcumin and/or IL-1β. We demonstrate that although curcumin alone does not have chondrogenic effects on MSCs, it inhibits IL-1β-induced activation of NF-κB, activation of caspase-3 and cyclooxygenase-2 in MSCs time and concentration dependently, as it does in chondrocytes. In IL-1β stimulated co-cultures, four-hour pre-treatment with curcumin significantly enhanced the production of collagen type II, cartilage specific proteoglycans (CSPGs), β1-integrin, as well as activating MAPKinase signaling and suppressing caspase-3 and cyclooxygenase-2. Curcumin treatment may help establish a microenvironment in which the effects of pro-inflammatory cytokines are antagonized, thus facilitating chondrogenesis of MSC-like progenitor cells in vivo. This strategy may support the regeneration of articular cartilage.

  7. The influence of scaffold microstructure on chondrogenic differentiation of mesenchymal stem cells

    International Nuclear Information System (INIS)

    Zhang, Jingjing; Ge, Zigang; Wu, Yingnan; Lee, Eng Hin; Thote, Tanushree; Yang, Zheng

    2014-01-01

    Different forms of biomaterials, including microspheres, sponges, hydrogels and nanofibres have been broadly used in cartilage regeneration; however, effects of internal structures of biomaterials on chondrogenesis of mesenchymal stem cells (MSCs) remain largely unexplored. Here we investigated the effect of physical microenvironments of sponges and hydrogels on chondrogenic differentiation of MSCs. MSCs, cultured in these two scaffold systems, were induced with TGF-β 3  in chondrogeneic differentiation medium and the chondrogenic differentiation was evaluated and compared after three weeks. MSCs in the sponges clustered with spindle morphologies, while they distributed homogenously with round morphologies in the hydrogel. The MSCs proliferated faster in the sponge compared to that in the hydrogel. Significantly higher glycosaminoglycan and collagen II were found in the sponges but not in the hydrogels. The different tissue formation ability of MSCs in these two systems could be attributed to the different metabolic requirements and the cellular events prerequisite in the chondrogenic process of MSCs. It is reasonable to conclude that sponges with relatively active microenvironments that facilitate cell–cell contacts and cell–matrix interaction are optimal for early stage of chondrogeneic differentiation. (paper)

  8. Brain-derived neurotrophic factor mediates estradiol-induced dendritic spine formation in hippocampal neurons

    Science.gov (United States)

    Murphy, Diane D.; Cole, Nelson B.; Segal, Menahem

    1998-01-01

    Dendritic spines are of major importance in information processing and memory formation in central neurons. Estradiol has been shown to induce an increase of dendritic spine density on hippocampal neurons in vivo and in vitro. The neurotrophin brain-derived neurotrophic factor (BDNF) recently has been implicated in neuronal maturation, plasticity, and regulation of GABAergic interneurons. We now demonstrate that estradiol down-regulates BDNF in cultured hippocampal neurons to 40% of control values within 24 hr of exposure. This, in turn, decreases inhibition and increases excitatory tone in pyramidal neurons, leading to a 2-fold increase in dendritic spine density. Exogenous BDNF blocks the effects of estradiol on spine formation, and BDNF depletion with a selective antisense oligonucleotide mimics the effects of estradiol. Addition of BDNF antibodies also increases spine density, and diazepam, which facilitates GABAergic neurotransmission, blocks estradiol-induced spine formation. These observations demonstrate a functional link between estradiol, BDNF as a potent regulator of GABAergic interneurons, and activity-dependent formation of dendritic spines in hippocampal neurons. PMID:9736750

  9. Studies on estradiol-2/4-hydroxylase activity in rat brain and liver

    International Nuclear Information System (INIS)

    Theron, C.N.

    1985-03-01

    A sensitive and specific radio-enzymatic assay was used to study estradiol-2/4-hydroxylase activity in rat liver microsomes and in microsomes obtained from 6 discrete brain areas of the rat. Kinetic parameters were determined for these enzyme activities. The effects of different P-450 inhibitors on estradiol-2/4-hydroxylase activity in brain and liver microsomes were also studied. In both organs these enzyme activities were found to be located mainly in the microsomal fraction and were inhibited by the 3 P-450 inhibitors tested. The hepatic estradiol-2/4-hydroxylase activity in adult male rats was significantly higher than that of females, but the enzyme activity in the brain did not exhibit a similar sex difference. Furthermore, estradiol-2/4-hydroxylase activity in rat liver was strongly induced by phenobarbitone treatment, but not in the brain. The phenobarbitone-induced activity in male and female rats exhibited significant kinetic differences. In female rats sexual maturation was associated with significant changes in the apparent Km of estradiol-2/4-hydroxylases in the liver and hypothalamus. Evidence was found that the in vitro estradiol-2/4-hydroxylase activity in rat brain and liver is due to more than one form of microsomal P-450. Kinetic studies showed important differences between the estradiol-2/4-hydroxylase activities in the hippocampus and hypothalamus. Significant differences in estradiol-2/4-hydroxylase activities were observed in the 6 brain areas studied, with the hippocampus showing the highest, and the hypothalamus the lowest activity at all developmental stages in both male and female rats

  10. Acute treatment with 17beta-estradiol attenuates astrocyte-astrocyte and astrocyte-neuron communication.

    Science.gov (United States)

    Rao, Shilpa P; Sikdar, Sujit Kumar

    2007-12-01

    Astrocytes are now recognized as dynamic signaling elements in the brain. Bidirectional communication between neurons and astrocytes involves integration of neuronal inputs by astrocytes and release of gliotransmitters that modulate neuronal excitability and synaptic transmission. The ovarian steroid hormone, 17beta-estradiol, in addition to its rapid actions on neuronal electrical activity can rapidly alter astrocyte intracellular calcium concentration ([Ca2+]i) through a membrane-associated estrogen receptor. Using calcium imaging and electrophysiological techniques, we investigated the functional consequences of acute treatment with estradiol on astrocyte-astrocyte and astrocyte-neuron communication in mixed hippocampal cultures. Mechanical stimulation of an astrocyte evoked a [Ca2+]i rise in the stimulated astrocyte, which propagated to the surrounding astrocytes as a [Ca2+]i wave. Following acute treatment with estradiol, the amplitude of the [Ca2+]i elevation in astrocytes around the stimulated astrocyte was attenuated. Further, estradiol inhibited the [Ca2+]i rise in individual astrocytes in response to the metabotropic glutamate receptor agonist, trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid. Mechanical stimulation of astrocytes induced [Ca2+]i elevations and electrophysiological responses in adjacent neurons. Estradiol rapidly attenuated the astrocyte-evoked glutamate-mediated [Ca2+]i rise and slow inward current in neurons. Also, the incidence of astrocyte-induced increase in spontaneous postsynaptic current frequency was reduced in the presence of estradiol. The effects of estradiol were stereo-specific and reversible following washout. These findings may indicate that the regulation of neuronal excitability and synaptic transmission by astrocytes is sensitive to rapid estradiol-mediated hormonal control. (c) 2007 Wiley-Liss, Inc.

  11. Estradiol RIA kit in clinical practice

    International Nuclear Information System (INIS)

    Friedrich, W.; Lisse, K.; Bienert, R.; Flentje, H.; Koerner, H.; Wilken, T.; Akademie der Wissenschaften der DDR, Berlin-Buch. Zentralinstitut fuer Isotopen- und Strahlenforschung)

    1985-01-01

    First clinical experience with a estradiol RIA kit developed in the Central Institute for Isotope- and Radiation Research is reported. The kit was used for the daily control of estradiol level in patients, which were treated within the program for in vitro fertilization and embryo transfer. The time of incubation could be shortened by means of a double antibody technique and by use of a precipitation mixture to 2 h. The intraassay variation is 9.2%, the interassay variation is 15.1%, the recovery rate is 94%. The sensitivity of the test (B 0 -3SD) is about 120 pmol/l. The estradiol RIA kit satisfies clinical requirements. (author)

  12. Micro-Topographies Promote Late Chondrogenic Differentiation Markers in the ATDC5 Cell Line

    NARCIS (Netherlands)

    Le, Bach Q.; Vasilevich, Aliaksei; Vermeulen, Steven; Hulshof, Frits; Stamatialis, Dimitrios F.; Van Blitterswijk, Clemens A.; De Boer, Jan

    2017-01-01

    Chemical and mechanical cues are well-established influencers of in vitro chondrogenic differentiation of ATDC5 cells. Here, we investigate the role of topographical cues in this differentiation process, a study not been explored before. Previously, using a library of surface micro-topographies we

  13. Characterization of lipidic markers of chondrogenic differentiation using mass spectrometry imaging.

    Science.gov (United States)

    Rocha, Beatriz; Cillero-Pastor, Berta; Eijkel, Gert; Bruinen, Anne L; Ruiz-Romero, Cristina; Heeren, Ron M A; Blanco, Francisco J

    2015-02-01

    Mesenchymal stem cells (MSC) are an interesting alternative for cell-based therapy of cartilage defects attributable to their capacity to differentiate toward chondrocytes in the process termed chondrogenesis. The metabolism of lipids has recently been associated with the modulation of chondrogenesis and also with the development of pathologies related to cartilage degeneration. Information about the distribution and modulation of lipids during chondrogenesis could provide a panel of putative chondrogenic markers. Thus, the discovery of new lipid chondrogenic markers could be highly valuable for improving MSC-based cartilage therapies. In this work, MS imaging was used to characterize the spatial distribution of lipids in human bone marrow MSCs during the first steps of chondrogenic differentiation. The analysis of MSC micromasses at days 2 and 14 of chondrogenesis by MALDI-MSI led to the identification of 20 different lipid species, including fatty acids, sphingolipids, and phospholipids. Phosphocholine, several sphingomyelins, and phosphatidylcholines were found to increase during the undifferentiated chondrogenic stage. A particularly detected lipid profile was verified by TOF secondary ion MS. Using this technology, a higher intensity of phosphocholine-related ions was observed in the peripheral region of the micromasses collected at day 14. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Development of a model system to analyze chondrogenic differentiation of mesenchymal stem cells

    Science.gov (United States)

    Ruedel, Anke; Hofmeister, Simone; Bosserhoff, Anja-Katrin

    2013-01-01

    High-density cell culture is widely used for the analysis of cartilage development of human mesenchymal stem cells (HMSCs) in vitro. Several cell culture systems, as micromass, pellet culture and alginate culture, are applied by groups in the field to induce chondrogenic differentiation of HMSCs. A draw back of all model systems is the high amount of cells necessary for the experiments. Further, handling of large experimental approaches is difficult due to culturing e.g. in 15 ml tubes. Therefore, we aimed to develop a new model system based on “hanging drop” cultures using 10 to 100 fold less cells. Here, we demonstrate that differentiation of chondrogenic cells was induced as previously shown in other model systems. Real time RT-PCR analysis demonstrated that Collagen type II and MIA/CD-RAP were upregulated during culturing whereas for induction of hypertrophic markers like Collagen type X and AP-2 epsilon treatment with TGF beta was needed. To further test the system, siRNA against Sox9 was used and effects on chondrogenic gene expression were evaluated. In summary, the hanging drop culture system was determined to be a promising tool for in vitro chondrogenic studies. PMID:24294400

  15. Micro-Topographies Promote Late Chondrogenic Differentiation Markers in the ATDC5 Cell Line.

    Science.gov (United States)

    Le, Bach Q; Vasilevich, Aliaksei; Vermeulen, Steven; Hulshof, Frits; Stamatialis, Dimitrios F; van Blitterswijk, Clemens A; de Boer, Jan

    2017-05-01

    Chemical and mechanical cues are well-established influencers of in vitro chondrogenic differentiation of ATDC5 cells. Here, we investigate the role of topographical cues in this differentiation process, a study not been explored before. Previously, using a library of surface micro-topographies we found some distinct patterns that induced alkaline phosphatase (ALP) production in human mesenchymal stromal cells. ALP is also a marker for hypertrophy, the end stage of chondrogenic differentiation preceding bone formation. Thus, we hypothesized that these patterns could influence end-stage chondrogenic differentiation of ATDC5 cells. In this study, we randomly selected seven topographies among the ALP influencing hits. Cells grown on these surfaces displayed varying nuclear shape and actin filament structure. When stimulated with insulin-transferrin-selenium (ITS) medium, nodule formation occurred and in some cases showed alignment to the topographical patterns. Gene expression analysis of cells growing on topographical surfaces in the presence of ITS medium revealed a downregulation of early markers and upregulation of late markers of chondrogenic differentiation compared to cells grown on a flat surface. In conclusion, we demonstrated that surface topography in addition to other cues can promote hypertrophic differentiation suitable for bone tissue engineering.

  16. Metabolic programming of mesenchymal stromal cells by oxygen tension directs chondrogenic cell fate

    NARCIS (Netherlands)

    Leijten, Jeroen Christianus Hermanus; Georgi, Nicole; Moreira Teixeira, Liliana; van Blitterswijk, Clemens; Post, Janine Nicole; Karperien, Hermanus Bernardus Johannes

    2014-01-01

    Actively steering the chondrogenic differentiation of mesenchymal stromal cells (MSCs) into either permanent cartilage or hypertrophic cartilage destined to be replaced by bone has not yet been possible. During limb development, the developing long bone is exposed to a concentration gradient of

  17. In vitro chondrogenic differentiation of human adipose-derived stem cells with silk scaffolds

    Directory of Open Access Journals (Sweden)

    Hyeon Joo Kim

    2012-12-01

    Full Text Available Human adipose-derived stem cells have shown chondrogenic differentiation potential in cartilage tissue engineering in combination with natural and synthetic biomaterials. In the present study, we hypothesized that porous aqueous-derived silk protein scaffolds would be suitable for chondrogenic differentiation of human adipose-derived stem cells. Human adipose-derived stem cells were cultured up to 6 weeks, and cell proliferation and chondrogenic differentiation were investigated and compared with those in conventional micromass culture. Cell proliferation, glycosaminoglycan, and collagen levels in aqueous-derived silk scaffolds were significantly higher than in micromass culture. Transcript levels of SOX9 and type II collagen were also upregulated in the cell–silk constructs at 6 weeks. Histological examination revealed that the pores of the silk scaffolds were filled with cells uniformly distributed. In addition, chondrocyte-specific lacunae formation was evident and distributed in the both groups. The results suggest the biodegradable and biocompatible three-dimensional aqueous-derived silk scaffolds provided an improved environment for chondrogenic differentiation compared to micromass culture.

  18. Gene expression profile in human induced pluripotent stem cells: Chondrogenic differentiation in vitro, part A

    Science.gov (United States)

    Suchorska, Wiktoria Maria; Augustyniak, Ewelina; Richter, Magdalena; Trzeciak, Tomasz

    2017-01-01

    Human induced pluripotent stem cells (hiPSCs) offer promise in regenerative medicine, however more data are required to improve understanding of key aspects of the cell differentiation process, including how specific chondrogenic processes affect the gene expression profile of chondrocyte-like cells and the relative value of cell differentiation markers. The main aims of the present study were as follows: To determine the gene expression profile of chondrogenic-like cells derived from hiPSCs cultured in mediums conditioned with HC-402-05a cells or supplemented with transforming growth factor β3 (TGF-β3), and to assess the relative utility of the most commonly used chondrogenic markers as indicators of cell differentiation. These issues are relevant with regard to the use of human fibroblasts in the reprogramming process to obtain hiPSCs. Human fibroblasts are derived from the mesoderm and thus share a wide range of properties with chondrocytes, which also originate from the mesenchyme. Thus, the exclusion of dedifferentiation instead of chondrogenic differentiation is crucial. The hiPSCs were obtained from human primary dermal fibroblasts during a reprogramming process. Two methods, both involving embryoid bodies (EB), were used to obtain chondrocytes from the hiPSCs: EBs formed in a chondrogenic medium supplemented with TGF-β3 (10 ng/ml) and EBs formed in a medium conditioned with growth factors from HC-402-05a cells. Based on immunofluorescence and reverse transcription-quantiative polymerase chain reaction analysis, the results indicated that hiPSCs have the capacity for effective chondrogenic differentiation, in particular cells differentiated in the HC-402-05a-conditioned medium, which present morphological features and markers that are characteristic of mature human chondrocytes. By contrast, cells differentiated in the presence of TGF-β3 may demonstrate hypertrophic characteristics. Several genes [paired box 9, sex determining region Y-box (SOX) 5, SOX6

  19. Hydrostatic pressure promotes the proliferation and osteogenic/chondrogenic differentiation of mesenchymal stem cells: The roles of RhoA and Rac1

    Directory of Open Access Journals (Sweden)

    Yin-Hua Zhao

    2015-05-01

    Full Text Available Our previous studies have shown that hydrostatic pressure can serve as an active regulator for bone marrow mesenchymal stem cells (BMSCs. The current work further investigates the roles of cytoskeletal regulatory proteins Ras homolog gene family member A (RhoA and Ras-related C3 botulinum toxin substrate 1 (Rac1 in hydrostatic pressure-related effects on BMSCs. Flow cytometry assays showed that the hydrostatic pressure promoted cell cycle initiation in a RhoA- and Rac1-dependent manner. Furthermore, fluorescence assays confirmed that RhoA played a positive and Rac1 displayed a negative role in the hydrostatic pressure-induced F-actin stress fiber assembly. Western blots suggested that RhoA and Rac1 play central roles in the pressure-inhibited ERK phosphorylation, and Rac1 but not RhoA was involved in the pressure-promoted JNK phosphorylation. Finally, real-time polymerase chain reaction (PCR experiments showed that pressure promoted the expression of osteogenic marker genes in BMSCs at an early stage of osteogenic differentiation through the up-regulation of RhoA activity. Additionally, the PCR results showed that pressure enhanced the expression of chondrogenic marker genes in BMSCs during chondrogenic differentiation via the up-regulation of Rac1 activity. Collectively, our results suggested that RhoA and Rac1 are critical to the pressure-induced proliferation and differentiation, the stress fiber assembly, and MAPK activation in BMSCs.

  20. Hydrostatic pressure promotes the proliferation and osteogenic/chondrogenic differentiation of mesenchymal stem cells: The roles of RhoA and Rac1.

    Science.gov (United States)

    Zhao, Yin-Hua; Lv, Xin; Liu, Yan-Li; Zhao, Ying; Li, Qiang; Chen, Yong-Jin; Zhang, Min

    2015-05-01

    Our previous studies have shown that hydrostatic pressure can serve as an active regulator for bone marrow mesenchymal stem cells (BMSCs). The current work further investigates the roles of cytoskeletal regulatory proteins Ras homolog gene family member A (RhoA) and Ras-related C3 botulinum toxin substrate 1 (Rac1) in hydrostatic pressure-related effects on BMSCs. Flow cytometry assays showed that the hydrostatic pressure promoted cell cycle initiation in a RhoA- and Rac1-dependent manner. Furthermore, fluorescence assays confirmed that RhoA played a positive and Rac1 displayed a negative role in the hydrostatic pressure-induced F-actin stress fiber assembly. Western blots suggested that RhoA and Rac1 play central roles in the pressure-inhibited ERK phosphorylation, and Rac1 but not RhoA was involved in the pressure-promoted JNK phosphorylation. Finally, real-time polymerase chain reaction (PCR) experiments showed that pressure promoted the expression of osteogenic marker genes in BMSCs at an early stage of osteogenic differentiation through the up-regulation of RhoA activity. Additionally, the PCR results showed that pressure enhanced the expression of chondrogenic marker genes in BMSCs during chondrogenic differentiation via the up-regulation of Rac1 activity. Collectively, our results suggested that RhoA and Rac1 are critical to the pressure-induced proliferation and differentiation, the stress fiber assembly, and MAPK activation in BMSCs. Copyright © 2015. Published by Elsevier B.V.

  1. Characterization of human adipose-derived stem cells and expression of chondrogenic genes during induction of cartilage differentiation

    Directory of Open Access Journals (Sweden)

    Adila A Hamid

    2012-01-01

    Full Text Available OBJECTIVES: Understanding the changes in chondrogenic gene expression that are involved in the differentiation of human adipose-derived stem cells to chondrogenic cells is important prior to using this approach for cartilage repair. The aims of the study were to characterize human adipose-derived stem cells and to examine chondrogenic gene expression after one, two, and three weeks of induction. MATERIALS AND METHODS: Human adipose-derived stem cells at passage 4 were evaluated by flow cytometry to examine the expression of surface markers. These adipose-derived stem cells were tested for adipogenic and osteogenic differentiation capacity. Ribonucleic acid was extracted from the cells for quantitative polymerase chain reaction analysis to determine the expression levels of chondrogenic genes after chondrogenic induction. RESULTS: Human adipose-derived stem cells were strongly positive for the mesenchymal markers CD90, CD73, CD44, CD9, and histocompatibility antigen and successfully differentiated into adipogenic and osteogenic lineages. The human adipose-derived stem cells aggregated and formed a dense matrix after chondrogenic induction. The expression of chondrogenic genes (collagen type II, aggrecan core protein, collagen type XI, COMP, and ELASTIN was significantly higher after the first week of induction. However, a significantly elevated expression of collagen type X was observed after three weeks of chondrogenic induction. CONCLUSION: Human adipose-derived stem cells retain stem cell characteristics after expansion in culture to passage 4 and serve as a feasible source of cells for cartilage regeneration. Chondrogenesis in human adiposederived stem cells was most prominent after one week of chondrogenic induction.

  2. Guanine nucleotide regulation of dopamine receptor agonist affinity states in rat estradiol-induced pituitary tumors

    Energy Technology Data Exchange (ETDEWEB)

    Di Paolo, T.; Falardeau, P.

    1987-08-31

    The authors have investigated dopamine (DA) receptor agonist high- and low-affinity states in female rate estradiol-induced prolactin (PRL)-secreting pituitary tumors and intact pituitary tissue. Estradiol treatment increased the anterior pituitary weight 9-fold and plasma prolactin levels 74-fold and these measures are correlated (R = 0.745, n = 73, p < 0.001). Competition for (/sup 3/H)-spiperone binding to the DA receptor by apomorphine was compared in normal and adenomatous pituitary tissue. The inhibition constants (Ki) and the proportions of the two apomorphine sites are unchanged in tumors compared to intact pituitary tissue. Guanosine 5'-(..beta..-..gamma..-imino)triphosphate (Gpp(NH)p) causes complete conversion of the high into low affinity dopaminergic agonist site in normal pituitary and in tumors. These results suggest that rats with primary estradiol-induced pituitary tumors have normal and functional DA receptors. 9 references, 2 tables.

  3. Guanine nucleotide regulation of dopamine receptor agonist affinity states in rat estradiol-induced pituitary tumors

    International Nuclear Information System (INIS)

    Di Paolo, T.; Falardeau, P.

    1987-01-01

    The authors have investigated dopamine (DA) receptor agonist high- and low-affinity states in female rate estradiol-induced prolactin (PRL)-secreting pituitary tumors and intact pituitary tissue. Estradiol treatment increased the anterior pituitary weight 9-fold and plasma prolactin levels 74-fold and these measures are correlated (R = 0.745, n = 73, p 3 H]-spiperone binding to the DA receptor by apomorphine was compared in normal and adenomatous pituitary tissue. The inhibition constants (Ki) and the proportions of the two apomorphine sites are unchanged in tumors compared to intact pituitary tissue. Guanosine 5'-[β-γ-imino]triphosphate (Gpp(NH)p) causes complete conversion of the high into low affinity dopaminergic agonist site in normal pituitary and in tumors. These results suggest that rats with primary estradiol-induced pituitary tumors have normal and functional DA receptors. 9 references, 2 tables

  4. Oleocanthal Modulates Estradiol-Induced Gene Expression Involving Estrogen Receptor α.

    Science.gov (United States)

    Keiler, Annekathrin Martina; Djiogue, Sefirin; Ehrhardt, Tino; Zierau, Oliver; Skaltsounis, Leandros; Halabalaki, Maria; Vollmer, Günter

    2015-09-01

    Oleocanthal is a bioactive compound from olive oil. It has attracted considerable attention as it is anti-inflammatory, antiproliferative, and has been shown to possess neuroprotective properties in vitro and in vivo. Delineated from its polyphenolic structure, the aim of this study was to characterize oleocanthal towards estrogenic properties. This might contribute to partly explain the beneficial effects described for the Mediterranean diet. Estrogenic properties of oleocanthal were assessed by different methods: a) stimulation of reporter gene activity in MVLN or RNDA cells either expressing estrogen receptor α or β, b) stimulation of luciferase reporter gene activity in U2OS osteosarcoma cells expressing estrogen receptor α or β, and c) elucidation of the impact on estradiol-induced gene expression in U2OS cells transduced with both estrogen receptors. Depending on the cell line origin, oleocanthal inhibited luciferase activity (MVLN, U2OS-estrogen receptor β) or weakly induced reporter gene activity at 10 µM in U2OS-estrogen receptor α cells. However, oleocanthal inhibited stimulation of luciferase activity by estradiol from both estrogen receptors. Oleocanthal, if given alone, did not stimulate gene expression in U2OS cells, but it significantly modulated the response of estradiol. Oleocanthal enhanced the effect of estradiol on the regulation of those genes, which are believed to be regulated through heterodimeric estrogen receptors. As the estrogenic response pattern of oleocanthal is rather unique, we compared the results obtained with oleacein. Oleocanthal binds to both estrogen receptors inducing estradiol-agonistic or antiagonistic effects depending on the cell line. Regarding regulation of gene expression in U2OS-estrogen receptor α/β cells, oleocanthal and oleacein enhanced estradiol-mediated regulation of heterodimer-regulated genes. Georg Thieme Verlag KG Stuttgart · New York.

  5. Neonatal androgenization of hypogonadal (hpg male mice does not abolish estradiol-induced FSH production and spermatogenesis

    Directory of Open Access Journals (Sweden)

    Kerr Jeffrey B

    2005-09-01

    Full Text Available Abstract Background Testicular development is arrested in the hypogonadal (hpg mouse due to a congenital deficiency in hypothalamic gonadotropin-releasing hormone (GnRH synthesis. Chronic treatment of male hpg mice with estradiol induces FSH synthesis and secretion, and causes testicular maturation and qualitatively normal spermatogenesis. As estradiol negative feedback normally inhibits FSH production in the male, this study tested whether this paradoxical response to estradiol in the male hpg mouse might be due to inadequate masculinisation or incomplete defeminization in the neonatal period. Previous studies have demonstrated that treatment of hpg mice with testosterone propionate in the immediate neonatal period is necessary to allow full reproductive behaviors to be expressed following suitable endocrine stimulation at adult ages. Methods Hpg mice were treated with 100 μg testosterone propionate or vehicle on postnatal day 2. At 35 days of age, subgroups of these mice were treated with silastic implants containing estradiol or cholesterol. Reproductive behavior was scored in tests with steroid-primed female mice, then testicular development was assessed histologically, and measures of pituitary FSH content made at 85 days of age. Results The neonatal testosterone propionate treatment successfully defeminized female litter mates, as revealed by impaired vaginal opening and deficiencies in lordosis behavior, and it allowed appropriate male reproductive behavior to be expressed in a proportion of the hpg males when tested at an adult age. However, neonatal androgen supplementation did not block or even reduce the subsequent actions of estradiol in increasing pituitary FSH content, nor did it affect the ability of estradiol to induce qualitatively normal spermatogenesis. Conclusion The ability of the hpg male to show a "female" neuroendocrine response to estradiol is not a result of inadequate androgenization during neonatal development, and

  6. Normal endometrial stromal cells regulate 17β-estradiol-induced epithelial-mesenchymal transition via slug and E-cadherin in endometrial adenocarcinoma cells in vitro.

    Science.gov (United States)

    Zhang, Hui; Li, Hongyan; Qi, Shasha; Liu, Zhao; Fu, Yibing; Li, Mingjiang; Zhao, Xingbo

    2017-01-01

    Stroma-tumor communication participates in the pathogenesis of endometrial carcinomas. In previous studies, we found that normal stromal cells inhibited the growth of endometrial carcinoma cells. Here, we investigated the role of normal stromal cells in the epithelial-mesenchymal transition (EMT) of endometrial carcinoma cells and explored the possible mechanism implied. We found that conditioned medium (CM) by normal endometrial stromal cells (NSC) reduced cell growth and induced cell apoptosis in Ishikawa cells. CM by NSC inhibited 17β-estradiol-induced cell growth and apoptosis decrease in Ishikawa cells. Moreover, CM by NSC inhibited the migration and invasion, and 17β-estradiol-induced migration and invasion in Ishikawa cells. Meanwhile, CM by NSC decreased Slug expression and 17β-estradiol-induced Slug expression, increased E-cadherin expression and abolished 17β-estradiol-induced E-cadherin reduction in Ishikawa cells. In conclusion, normal stromal factors can inhibit 17β-estradiol-induced cell proliferation and apoptosis inhibition, and abolished 17β-estradiol-induced EMT in endometrial cancer cell via regulating E-cadherin and Slug expression.

  7. Paradoxical action of fulvestrant in estradiol-induced regression of tamoxifen-stimulated breast cancer.

    Science.gov (United States)

    Osipo, Clodia; Gajdos, Csaba; Liu, Hong; Chen, Bin; Jordan, V Craig

    2003-11-05

    Long-term tamoxifen treatment of breast cancer can result in tamoxifen-stimulated breast cancer, in which estrogen inhibits tumor growth after tamoxifen withdrawal. We investigated the molecular mechanism(s) of estradiol-induced tumor regression by using an in vivo model of tamoxifen-stimulated human breast cancer. Growth of parental estradiol-stimulated MCF-7E2 and long-term tamoxifen-stimulated MCF-7TAMLT xenografts in athymic mice was measured during treatment with vehicle, estradiol, estradiol plus tamoxifen, tamoxifen alone, estradiol plus fulvestrant, or fulvestrant alone. Apoptosis was detected by the terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. Protein expression was assessed by western blot analysis. mRNA expression was assessed by real-time reverse transcription-polymerase chain reaction. All statistical tests were two-sided. MCF-7E2 tumor growth was stimulated by estradiol (cross-sectional area at week 13 = 1.06 cm2, 95% confidence interval [CI] = 0.82 to 1.30 cm2; Pestradiol-induced regression to 0.18 cm2 (95% CI = 0.15 to 0.21 cm2; P<.001), and tamoxifen or estradiol plus fulvestrant enhanced tumor growth to 1.00 cm2 (95% CI = 0.88 to 1.22 cm2). Estradiol increased the number of apoptotic cells in tumors by 23% (95% CI = 20% to 26%; P<.001) compared with all other treatments, decreased estrogen receptor alpha(ERalpha) protein expression, increased the expression of Fas mRNA and protein, decreased the expression of HER2/neu mRNA and protein and nuclear factor kappaB (NF-kappaB) protein but did not affect Fas ligand protein expression compared with control. Paradoxically, fulvestrant reversed this effect and stimulated MCF-7TAMLT tumor growth apparently through ERalpha-mediated regulation of Fas, HER2/neu, and NF-kappaB. Physiologic levels of estradiol induced regression of tamoxifen-stimulated breast cancer tumors, apparently by inducing the death receptor Fas and suppressing the antiapoptotic

  8. Estradiol suppresses tissue androgens and prostate cancer growth in castration resistant prostate cancer

    International Nuclear Information System (INIS)

    Montgomery, Bruce; Nelson, Peter S; Vessella, Robert; Kalhorn, Tom; Hess, David; Corey, Eva

    2010-01-01

    Estrogens suppress tumor growth in prostate cancer which progresses despite anorchid serum androgen levels, termed castration resistant prostate cancers (CRPC), although the mechanisms are unclear. We hypothesize that estrogen inhibits CRPC in anorchid animals by suppressing tumoral androgens, an effect independent of the estrogen receptor. The human CRPC xenograft LuCaP 35V was implanted into orchiectomized male SCID mice and established tumors were treated with placebo, 17β-estradiol or 17β-estradiol and estrogen receptor antagonist ICI 182,780. Effects of 17β-estradiol on tumor growth were evaluated and tissue testosterone (T) and dihydrotestosterone (DHT) evaluated by mass spectrometry. Treatment of LuCaP 35V with 17β-estradiol slowed tumor growth compared to controls (tumor volume at day 21: 785 ± 81 mm 3 vs. 1195 ± 84 mm 3 , p = 0.002). Survival was also significantly improved in animals treated with 17β-estradiol (p = 0.03). The addition of the estrogen receptor antagonist ICI 182,780 did not significantly change survival or growth. 17β-estradiol in the presence and absence of ICI 182,780 suppressed tumor testosterone (T) and dihydrotestosterone (DHT) as assayed by mass spectrometry. Tissue androgens in placebo treated LuCaP 35V xenografts were; T = 0.71 ± 0.28 pg/mg and DHT = 1.73 ± 0.36 pg/mg. In 17β-estradiol treated LuCaP35V xenografts the tissue androgens were, T = 0.20 ± 0.10 pg/mg and DHT = 0.15 ± 0.15 pg/mg, (p < 0.001 vs. controls). Levels of T and DHT in control liver tissue were < 0.2 pg/mg. CRPC in anorchid animals maintains tumoral androgen levels despite castration. 17β-estradiol significantly suppressed tumor T and DHT and inhibits growth of CRPC in an estrogen receptor independent manner. The ability to manipulate tumoral androgens will be critical in the development and testing of agents targeting CRPC through tissue steroidogenesis

  9. Surface Markers for Chondrogenic Determination: A Highlight of Synovium-Derived Stem Cells

    Directory of Open Access Journals (Sweden)

    Douglas D. Campbell

    2012-11-01

    Full Text Available Cartilage tissue engineering is a promising field in regenerative medicine that can provide substantial relief to people suffering from degenerative cartilage disease. Current research shows the greatest chondrogenic potential for healthy articular cartilage growth with minimal hypertrophic differentiation to be from mesenchymal stem cells (MSCs of synovial origin. These stem cells have the capacity for differentiation into multiple cell lineages related to mesenchymal tissue; however, evidence exists for cell surface markers that specify a greater potential for chondrogenesis than other differentiation fates. This review will examine relevant literature to summarize the chondrogenic differentiation capacities of tested synovium-derived stem cell (SDSC surface markers, along with a discussion about various other markers that may hold potential, yet require further investigation. With this information, a potential clinical benefit exists to develop a screening system for SDSCs that will produce the healthiest articular cartilage possible.

  10. Chondrogenic potential of physically treated bovine cartilage matrix derived porous scaffolds on human dermal fibroblast cells.

    Science.gov (United States)

    Moradi, Ali; Ataollahi, Forough; Sayar, Katayoun; Pramanik, Sumit; Chong, Pan-Pan; Khalil, Alizan Abdul; Kamarul, Tunku; Pingguan-Murphy, Belinda

    2016-01-01

    Extracellular matrices have drawn attention in tissue engineering as potential biomaterials for scaffold fabrication because of their bioactive components. Noninvasive techniques of scaffold fabrication and cross-linking treatments are believed to maintain the integrity of bioactive molecules while providing proper architectural and mechanical properties. Cartilage matrix derived scaffolds are designed to support the maintenance of chondrocytes and provide proper signals for differentiation of chondroinducible cells. Chondroinductive potential of bovine articular cartilage matrix derived porous scaffolds on human dermal fibroblasts and the effect of scaffold shrinkage on chondrogenesis were investigated. An increase in sulfated glycosaminoglycans production along with upregulation of chondrogenic genes confirmed that physically treated cartilage matrix derived scaffolds have chondrogenic potential on human dermal fibroblasts. © 2015 Wiley Periodicals, Inc.

  11. Antioxidant protection of LDL by physiological concentrations of 17 beta-estradiol. Requirement for estradiol modification.

    Science.gov (United States)

    Shwaery, G T; Vita, J A; Keaney, J F

    1997-03-18

    Exposure to estrogens reduces the risk for coronary artery disease and associated clinical events; however, the mechanisms responsible for these observations are not clear. Supraphysiological levels of estrogens act as antioxidants in vitro, limiting oxidation of low-density lipoprotein (LDL), an event implicated in atherogenesis. We investigated the conditions under which physiological concentrations of 17 beta-estradiol (E2) inhibit oxidative modification of LDL. Plasma incubated with E2 (0.1 to 100 nmol/L) for 4 hours yielded LDL that demonstrated a dose-related increase in resistance to oxidation by Cu2+ as measured by conjugated diene formation. This effect was dependent on plasma, because incubation of isolated LDL with E2 at these concentrations in buffered saline produced no effect on Cu(2+)-mediated oxidation. Incubation of plasma with E2 had no effect on LDL alpha-tocopherol content or cholesteryl ester hydroperoxide formation during the 4-hour incubation. Plasma incubation with [3H]E2 was associated with dose-dependent association of 3H with LDL. High-performance liquid chromatographic analysis of LDL derived from plasma incubated with [3H]E2 indicated that the majority of the associated species were not detectable as authentic E2 but as nonpolar forms of E2 that were susceptible to base hydrolysis consistent with fatty acid esterification of E2. Plasma-mediated association of E2 and subsequent antioxidant protection was inhibited by 5,5'-dithiobis(2-nitrobenzoic acid), an inhibitor of plasma acyltransferase activity. Exposure of LDL to physiological levels of E2 in a plasma milieu is associated with enhanced resistance to Cu(2+)-mediated oxidation and incorporation of E2 derivatives into LDL. This antioxidant capacity may be another means by which E2 limits coronary artery disease in women.

  12. Evaluation of Insulin Medium or Chondrogenic Medium on Proliferation and Chondrogenesis of ATDC5 Cells

    OpenAIRE

    Yao, Yongchang; Zhai, Zhichen; Wang, Yingjun

    2014-01-01

    Background. The ATDC5 cell line is regarded as an excellent cell model for chondrogenesis. In most studies with ATDC5 cells, insulin medium (IM) was used to induce chondrogenesis while chondrogenic medium (CM), which was usually applied in chondrogenesis of mesenchymal stem cells (MSCs), was rarely used for ATDC5 cells. This study was mainly designed to investigate the effect of IM, CM, and growth medium (GM) on chondrogenesis of ATDC5 cells. Methods. ATDC5 cells were, respectively, cultured ...

  13. Nicotine-induced chondrogenic differentiation of human bone marrow stromal cells in vitro.

    Science.gov (United States)

    Ying, Xiaozhou; Zhang, Wei; Cheng, Shaowen; Nie, Pengfei; Cheng, Xiaojie; Shen, Yue; Wang, Wei; Xue, Enxing; Chen, Qingyu; Kou, Dongquan; Peng, Lei; Zhang, Yu; Lu, Chuanzhu

    2012-11-01

    Nicotine has been reported that it has a dose-dependent effect on matrix mineralization by human bone marrow cells. However, there is no relevant research concerning on chondrogenic differentiation potential of bone marrow stromal stem cells (BMSCs) treated with nicotine in vitro. The aims of the study were to examine the effects of nicotine (0, 10(-7), 10(-6) and 10(-5) M) on the proliferation and chondrogenic differentiation of BMSCs from three healthy donors in vitro. BMSCs proliferation was analyzed by CCK8 assay and real-time polymerase chain reaction was used to assay the expression of type II collagen, aggrecan, type I collagen and type X collagen. The proteoglycan content was stained by Alcian blue, and the sulfated glycosaminoglycan (sGAG) content of BMSCs was quantified spectrofluorometrically using dimethylmethylene blue. The cell viability was not significantly impaired until up to a concentration of 10(-5) M nicotine. Nicotine promoted the proliferation and enhanced the expression of type II collagen at the level up to 10(-6) M (P < 0.05). The expression of aggrecan was reduced at the concentration of 10(-5) M nicotine at day 14 (P < 0.05), and there was no significant difference in aggrecan gene expression at 10(-7) and 10(-6) M nicotine levels compared to control group (n.s.). Also the fibroblastic and hypertrophic gene expressions were down-regulated in the chondrogenic medium with 10(-7)-10(-5) M nicotine (P < 0.05). It was implied that local application of nicotine at an appropriate concentration may be a promising approach for enhancing chondrogenic differentiation capacity of BMSCs in cell-based cartilage tissue engineering. Also these results indicate that nicotine maybe a potentially useful drug for the treatment of Osteoarthritis.

  14. Novel biologically-inspired rosette nanotube PLLA scaffolds for improving human mesenchymal stem cell chondrogenic differentiation

    International Nuclear Information System (INIS)

    Childs, Allie; Castro, Nathan J; Zhang, Lijie Grace; Hemraz, Usha D; Fenniri, Hicham

    2013-01-01

    Cartilage defects are a persistent issue in orthopedic tissue engineering where acute and chronic tissue damage stemming from osteoarthritis, trauma, and sport injuries, present a common and serious clinical problem. Unlike bone, cartilage repair continues to be largely intractable due to the tissue's inherently poor regenerative capacity. Thus, the objective of this study is to design a novel tissue engineered nanostructured cartilage scaffold via biologically-inspired self-assembling rosette nanotubes (RNTs) and biocompatible non-woven poly (l-lactic acid) (PLLA) for enhanced human bone marrow mesenchymal stem cell (hMSC) chondrogenic differentiation. Specifically, RNTs are a new class of biomimetic supramolecular nanomaterial obtained through the self-assembly of low-molecular-weight modified guanine/cytosine DNA base hybrids (the G∧C motif) in an aqueous environment. In this study, we synthesized a novel twin G∧C-based RNT (TB-RGDSK) functionalized with cell-favorable arginine–glycine–aspartic acid–serine–lysine (RGDSK) integrin binding peptide and a twin G∧C based RNT with an aminobutane linker molecule (TBL). hMSC adhesion, proliferation and chondrogenic differentiation were evaluated in vitro in scaffold groups consisting of biocompatible PLLA with TBL, 1:9 TB-RGDSK:TBL, and TB-RGDSK, respectively. Our results show that RNTs can remarkably increase total glycosaminoglycan, collagen, and protein production when compared to PLLA controls without nanotubes. Furthermore, the TB-RGDSK with 100% well-organized RGDSK peptides achieved the highest chondrogenic differentiation of hMSCs. The current in vitro study illustrated that RNT nanotopography and surface chemistry played an important role in enhancing hMSC chondrogenic differentiation thus making them promising for cartilage regeneration. (paper)

  15. Effects of BIO on proliferation and chondrogenic differentiation of mouse marrow derived mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Nasrin Fallah

    2013-06-01

    Full Text Available In vitroexpansion of mesenchymal stem cell (MSCs into large number is necessary fortheir application in cell-based treatment of articular cartilage defects. On the other hand,some studies have indicated that BIO (6-Bromoindirubin-3-Oxime possesses mitogeniceffects on cell culture. The objective of the present study was to examine the effect of BIO onin vitro expansion and chondrogenic differentiation of mouse marrow-derived MSCs. Theculture was established using bone marrow tissue obtained from 10 NMRI mice. MSC natureof the isolated cells was verified according to the minimal criteria proposed for MSC.Passaged-3 cells were seeded in 24-well culture plates and treated by 0.05, 0.01, 0.1, 1.0 and1.5 μM BIO forsevendays. The culture without BIO was taken as the control. At the end ofcultivation period, the cultures were examinedfor viable cell number which was then used tocalculate population doubling time (PDT. The BIO with higher proliferation-promoting effectwas investigated for its chondrogenic effect on MSC culture. There was significantly moreviable cells at the cultures treated by 0.1 μM BIO. At this culture the cells tended to doubletheir population in rapid rate (each 43.07 hr than the cells treated with the other BIOconcentrations (p< 0.05. Interestingly treatment of MSC chondrogenic culture with 0.1 μMBIO ledto the up-regulation of cartilage specific genes including aggrecan, collagen II andSox9. In conclusion BIO at 0.1 μM could enhance mouse MSC in vitro proliferation as well astheir chondrogenic differentiation. These findings would be of great importance for the fieldof regenerative medicine.

  16. Platelet lysate induces chondrogenic differentiation of umbilical cord-derived mesenchymal stem cells.

    Science.gov (United States)

    Hassan, Ghmkin; Bahjat, Mohammad; Kasem, Issam; Soukkarieh, Chadi; Aljamali, Majd

    2018-01-01

    Articular cartilage has a poor capacity for self-repair, and thus still presents a major challenge in orthopedics. Mesenchymal stem cells (MSCs) are multipotent stem cells with the potential to differentiate into chondrocytes in the presence of transforming growth factor beta (TGF-β). Platelet lysate (PL) contains a relatively large number of growth factors, including TGF-β, and has been shown to ameliorate cartilage repair. Here, we investigated the ability of PL to direct chondrogenic differentiation of MSCs along with other standard differentiation components in a pellet culture system. We isolated and expanded MSCs from human umbilical cords using a PL-supplemented medium and characterized the cells based on immunophenotype and potential for differentiation to adipocytes and osteocytes. We further cultured MSCs as pellets in a chondrogenic-differentiation medium supplemented with PL. After 21 days, the pellets were processed for histological analysis and stained with alician blue and acridine orange. The expression of SOX9 was investigated using RT-PCR. MSCs maintained their stemness characteristics in the PL-supplemented medium. However, the distribution of cells in the pellets cultured in the PL-supplemented chondrogenic differentiation medium had a greater similarity to cartilage tissue-derived chondrocytes than to the negative control. The intense alician blue staining indicated an increased production of mucopolysaccharides in the differentiated pellets, which also showed elevated expression of SOX9 . Our data suggest that MSCs could be differentiated to chondrocytes in the presence of PL and absence of exogenous TGF-β. Further research needs to be conducted to understand the exact role and potential of PL in chondrogenic differentiation and chondrocyte regeneration.

  17. Generation of Col2a1-EGFP iPS cells for monitoring chondrogenic differentiation.

    Directory of Open Access Journals (Sweden)

    Taku Saito

    Full Text Available Induced pluripotent stem cells (iPSC are a promising cell source for cartilage regenerative medicine; however, the methods for chondrocyte induction from iPSC are currently developing and not yet sufficient for clinical application. Here, we report the establishment of a fluorescent indicator system for monitoring chondrogenic differentiation from iPSC to simplify screening for effective factors that induce chondrocytes from iPSC. We generated iPSC from embryonic fibroblasts of Col2a1-EGFP transgenic mice by retroviral transduction of Oct4, Sox2, Klf4, and c-Myc. Among the 30 clones of Col2a1-EGFP iPSC we established, two clones showed high expression levels of embryonic stem cell (ESC marker genes, similar to control ESC. A teratoma formation assay showed that the two clones were pluripotent and differentiated into cell types from all three germ layers. The fluorescent signal was observed during chondrogenic differentiation of the two clones concomitant with the increase in chondrocyte marker expression. In conclusion, Col2a1-EGFP iPSC are useful for monitoring chondrogenic differentiation and will contribute to research in cartilage regenerative medicine.

  18. Estrogen inhibits chloride secretion caused by cholera and Escherichia coli enterotoxins in female rat distal colon.

    OpenAIRE

    Alzamora, Rodrigo; O'Mahony, Fiona; Harvey, Brian J

    2011-01-01

    Excessive Cl(-) secretion is the driving force for secretory diarrhea. 17β-Estradiol has been shown to inhibit Cl(-) secretion in rat distal colon through a nongenomic pathway. We examined whether 17β-estradiol inhibits Cl(-) secretion in an animal model of secretory diarrhea and the downstream effectors involved. The effect of 17β-estradiol on cholera toxin and heat-stable enterotoxin induced Cl(-) secretion in rat colonic mucosal sheets was studied by current-voltage clamping. Selective per...

  19. Ethinyl Estradiol and Etonogestrel Vaginal Ring

    Science.gov (United States)

    ... or infection of the vagina white or yellow vaginal discharge vaginal bleeding or spotting when it is not time ... Follow your doctor's directions for examining your breasts; report any lumps ... and ethinyl estradiol vaginal ring.Do not let anyone else use your ...

  20. Biological activity and binding of estradiol to SK-Mel 23 human melanoma cells

    Directory of Open Access Journals (Sweden)

    Sarti M.S.M.V.

    2004-01-01

    Full Text Available Patients expressing estradiol receptors in melanoma cells have been reported to have a better prognosis. We therefore decided to investigate the in vitro effects of ß-estradiol and tamoxifen on the growth and tyrosinase activity of SK-Mel 23 human melanoma cells. Twenty-four-hour treatment with 0.4 nM ß-estradiol inhibited cell proliferation in 30% (0.70 ± 0.03 x 10(5 cells and increased tyrosinase activity in 50% (7130.5 ± 376.5 cpm/10(5 cells, as compared to untreated cells (1.0 ± 0.05 x 10(5 cells and 4769 ± 25.5 cpm/10(5 cells, respectively. Both responses were completely (100% blocked by 1 µM tamoxifen. Higher concentrations (up to 1.6 nM or longer treatments (up to 72 h did not result in a larger effect of the hormone on proliferation or tyrosinase activity. Competition binding assays demonstrated the presence of binding sites to [2,4,6,7-³H]-ß-estradiol, and that the tritiated analogue was displaced by the unlabeled hormone (1 nM to 100 µM, Kd = 0.14 µM, maximal displacement of 93% or by 10 µM tamoxifen (displacement of 60%. ß-estradiol also increased the phosphorylated state of two proteins of 16 and 46 kDa, after 4-h treatment, as determined by Western blot. The absorbance of each band was 1.9- and 4-fold the controls, respectively, as determined with Image-Pro Plus software. Shorter incubation periods with ß-estradiol did not enhance phosporylation; after 6-h treatment with the hormone, the two proteins returned to the control phosphorylation levels. The growth inhibition promoted by estradiol may explain the better prognosis of melanoma-bearing women as compared to men, and open new perspectives for drug therapy.

  1. An electromagnetic compressive force by cell exciter stimulates chondrogenic differentiation of bone marrow-derived mesenchymal stem cells.

    Science.gov (United States)

    Park, Sang-Hyug; Sim, Woo Young; Park, Sin Wook; Yang, Sang Sik; Choi, Byung Hyune; Park, So Ra; Park, Kwideok; Min, Byoung-Hyun

    2006-11-01

    In this study, we present a biological micro-electromechanical system and its application to the chondrogenic differentiation of rabbit bone marrow-derived mesenchymal stem cells (MSCs). Actuated by an electromagnetic force, the micro cell exciter was designed to deliver a cyclic compressive load (CCL) with various magnitudes. Two major parts in the system are an actuator and a cartridge-type chamber. The former has a permanent magnet and coil, and the latter is equipped with 7 sample dishes and 7 metal caps. Mixed with a 2.4% alginate solution, the alginate/MSC layers were positioned in the sample dishes; the caps contained chondrogenic defined medium without transforming growth factor-beta (TGF-beta). Once powered, the actuator coil-derived electromagnetic force pulled the metal caps down, compressing the samples. The cyclic load was given at 1-Hz frequency for 10 min twice a day. Samples in the dishes without a cap served as a control. The samples were analyzed at 3, 5, and 7 days after stimulation for cell viability, biochemical assays, histologic features, immunohistochemistry, and gene expression of the chondrogenic markers. Applied to the alginate/MSC layer, the CCL system enhanced the synthesis of cartilage-specific matrix proteins and the chondrogenic markers, such as aggrecan, type II collagen, and Sox9. We found that the micromechanically exerted CCL by the cell exciter was very effective in enhancing the chondrogenic differentiation of MSCs, even without using exogenous TGF-beta.

  2. Attenuation of chondrogenic transformation in vascular smooth muscle by dietary quercetin in the MGP-deficient mouse model.

    Directory of Open Access Journals (Sweden)

    Kelly E Beazley

    Full Text Available Cartilaginous metaplasia of vascular smooth muscle (VSM is characteristic for arterial calcification in diabetes and uremia and in the background of genetic alterations in matrix Gla protein (MGP. A better understanding of the molecular details of this process is critical for the development of novel therapeutic approaches to VSM transformation and arterial calcification.This study aimed to identify the effects of bioflavonoid quercetin on chondrogenic transformation and calcification of VSM in the MGP-null mouse model and upon TGF-β3 stimulation in vitro, and to characterize the associated alterations in cell signaling.Molecular analysis revealed activation of β-catenin signaling in cartilaginous metaplasia in Mgp-/- aortae in vivo and during chondrogenic transformation of VSMCs in vitro. Quercetin intercepted chondrogenic transformation of VSM and blocked activation of β-catenin both in vivo and in vitro. Although dietary quercetin drastically attenuated calcifying cartilaginous metaplasia in Mgp-/- animals, approximately one-half of total vascular calcium mineral remained as depositions along elastic lamellae.Quercetin is potent in preventing VSM chondrogenic transformation caused by diverse stimuli. Combined with the demonstrated efficiency of dietary quercetin in preventing ectopic chondrogenesis in the MGP-null vasculature, these findings indicate a potentially broad therapeutic applicability of this safe for human consumption bioflavonoid in the therapy of cardiovascular conditions linked to cartilaginous metaplasia of VSM. Elastocalcinosis is a major component of MGP-null vascular disease and is controlled by a mechanism different from chondrogenic transformation of VSM and not sensitive to quercetin.

  3. PDK2 promotes chondrogenic differentiation of mesenchymal stem cells by upregulation of Sox6 and activation of JNK/MAPK/ERK pathway

    Directory of Open Access Journals (Sweden)

    H. Wang

    Full Text Available This study was undertaken to clarify the role and mechanism of pyruvate dehydrogenase kinase isoform 2 (PDK2 in chondrogenic differentiation of mesenchymal stem cells (MSCs. MSCs were isolated from femurs and tibias of Sprague-Dawley rats, weighing 300-400 g (5 females and 5 males. Overexpression and knockdown of PDK2 were transfected into MSCs and then cell viability, adhesion and migration were assessed. Additionally, the roles of aberrant PDK2 in chondrogenesis markers SRY-related high mobility group-box 6 (Sox6, type ΙΙ procollagen gene (COL2A1, cartilage oligomeric matrix protein (COMP, aggrecan (AGC1, type ΙX procollagen gene (COL9A2 and collagen type 1 alpha 1 (COL1A1 were measured by quantitative reverse-transcription polymerase chain reaction (qRT-PCR. The expressions of c-Jun N-terminal kinase (JNK, p38 mitogen-activated protein kinase (MAPK and extracellular regulated protein kinase (ERK were measured. Overexpressing PDK2 promoted cell viability, adhesion and inhibited cell migration in MSCs (all P<0.05. qRT-PCR assay showed a potent increase in the mRNA expressions of all chondrogenesis markers in response to overexpressing PDK2 (P<0.01 or P<0.05. PDK2 overexpression also induced a significant accumulation in mRNA and protein expressions of JNK, p38MAPK and ERK in MSCs compared to the control (P<0.01 or P<0.05. Meanwhile, silencing PDK2 exerted the opposite effects on MSCs. This study shows a preliminary positive role and potential mechanisms of PDK2 in chondrogenic differentiation of MSCs. It lays the theoretical groundwork for uncovering the functions of PDK2 and provides a promising basis for repairing cartilage lesions in osteoarthritis.

  4. Characterization of Chondrogenic Gene Expression and Cartilage Phenotype Differentiation in Human Breast Adipose-Derived Stem Cells Promoted by Ginsenoside Rg1 In Vitro

    Directory of Open Access Journals (Sweden)

    Fang-Tian Xu

    2015-11-01

    Full Text Available Background/Aims: Investigating and understanding chondrogenic gene expression during the differentiation of human breast adipose-derived stem cells (HBASCs into chondrogenic cells is a prerequisite for the application of this approach for cartilage repair and regeneration. In this study, we aim to characterize HBASCs and to examine chondrogenic gene expression in chondrogenic inductive culture medium containing ginsenoside Rg1. Methods: Human breast adipose-derived stem cells at passage 3 were evaluated based on specific cell markers and their multilineage differentiation capacity. Cultured HBASCs were treated either with basic chondrogenic inductive conditioned medium alone (group A, control or with basic chondrogenic inductive medium plus 10 µg/ml (group B, 50 µg/ml (group C, or 100µg/ml ginsenoside Rg1 (group D. Cell proliferation was assessed using the CCK-8 assay for a period of 9 days. Two weeks after induction, the expression of chondrogenic genes (collagen type II, collagen type XI, ACP, COMP and ELASTIN was determined using real-time PCR in all groups. Results: The different concentrations of ginsenoside Rg1 that were added to the basic chondrogenic inductive culture medium promoted the proliferation of HBASCs at earlier stages (groups B, C, and D but resulted in chondrogenic phenotype differentiation and higher mRNA expression of collagen type II (CO-II, collagen type XI (CO-XI, acid phosphatase (ACP, cartilage oligomeric matrix protein (COMP and ELASTIN compared with the control (group A at later stages. The results reveal an obvious positive dose-effect relationship between ginsenoside Rg1 and the proliferation and chondrogenic phenotype differentiation of HBASCs in vitro. Conclusions: Human breast adipose-derived stem cells retain stem cell characteristics after expansion in culture through passage 3 and serve as a feasible source of cells for cartilage regeneration in vitro. Chondrogenesis in HBASCs was found to be prominent

  5. Hydrostatic pressure acts to stabilise a chondrogenic phenotype in porcine joint tissue derived stem cells

    Directory of Open Access Journals (Sweden)

    T Vinardell

    2012-02-01

    Full Text Available Hydrostatic pressure (HP is a key component of the in vivo joint environment and has been shown to enhance chondrogenesis of stem cells. The objective of this study was to investigate the interaction between HP and TGF-β3 on both the initiation and maintenance of a chondrogenic phenotype for joint tissue derived stem cells. Pellets generated from porcine chondrocytes (CCs, synovial membrane derived stem cells (SDSCs and infrapatellar fat pad derived stem cells (FPSCs were subjected to 10 MPa of cyclic HP (4 h/day and different concentrations of TGF-β3 (0, 1 and 10 ng/mL for 14 days. CCs and stem cells were observed to respond differentially to both HP and TGF-β3 stimulation. HP in the absence of TGF-β3 did not induce robust chondrogenic differentiation of stem cells. At low concentrations of TGF-β3 (1 ng/mL, HP acted to enhance chondrogenesis of both SDSCs and FPSCs, as evident by a 3-fold increase in Sox9 expression and a significant increase in glycosaminoglycan accumulation. In contrast, HP had no effect on cartilage-specific matrix synthesis at higher concentrations of TGF-β3 (10 ng/mL. Critically, HP appears to play a key role in the maintenance of a chondrogenic phenotype, as evident by a down-regulation of the hypertrophic markers type X collagen and Indian hedgehog in SDSCs irrespective of the cytokine concentration. In the context of stem cell based therapies for cartilage repair, this study demonstrates the importance of considering how joint specific environmental factors interact to regulate not only the initiation of chondrogenesis, but also the development of a stable hyaline-like repair tissue.

  6. Paracrine and autocrine signals promoting full chondrogenic differentiation of a mesoblastic cell line.

    Science.gov (United States)

    Locker, Morgane; Kellermann, Odile; Boucquey, Marie; Khun, Huot; Huerre, Michel; Poliard, Anne

    2004-01-01

    The pluripotent mesoblastic C1 cell line was used under serum-free culture conditions to investigate how paracrine and autocrine signals cooperate to drive chondrogenesis. Sequential addition of two systemic hormones, dexamethasone and triiodothyronine, permits full chondrogenic differentiation. The cell intrinsic activation of the BMP signaling pathway and Sox9 expression occurring on mesoblastic condensation is insufficient for recruitment of the progenitors. Dexamethasone-dependent Sox9 upregulation is essential for chondrogenesis. Differentiation of lineage stem cells relies on cell autonomous regulations modulated by external signals. We used the pluripotent mesoblastic C1 cell line under serum-free culture conditions to investigate how paracrine and autocrine signals cooperate to induce differentiation of a precursor clone along the chondrogenic lineage. C1 cells, cultured as aggregates, were induced toward chondrogenesis by addition of 10(-7) M dexamethasone in serum-free medium. After 30 days, dexamethasone was replaced by 10 nM triiodothyronine to promote final hypertrophic conversion. Mature and hypertrophic phenotypes were characterized by immunocytochemistry using specific antibodies against types II and X collagens, respectively. Type II collagen, bone morphogenetic proteins (BMPs), BMP receptors, Smads, and Sox9 expression were monitored by reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blot, and/or Western blot analysis. Once C1 cells have formed nodules, sequential addition of two systemic hormones is sufficient to promote full chondrogenic differentiation. In response to dexamethasone, nearly 100% of the C1 precursors engage in chondrogenesis and convert within 30 days into mature chondrocytes, which triggers a typical cartilage matrix. On day 25, a switch in type II procollagen mRNA splicing acted as a limiting step in the acquisition of the mature chondrocyte phenotype. On day 30, substitution of dexamethasone with

  7. Chondrogenic differentiation of ATDC5-cells under the influence of Mg and Mg alloy degradation

    Energy Technology Data Exchange (ETDEWEB)

    Martinez Sanchez, Adela H., E-mail: adela.martinez@hzg.de [Helmholtz-Zentrum Geesthacht, Institute of Material Research, Department for Structural Research on Macromolecules, Geesthacht (Germany); Feyerabend, Frank; Laipple, Daniel; Willumeit-Römer, Regine [Helmholtz-Zentrum Geesthacht, Institute of Material Research, Department for Structural Research on Macromolecules, Geesthacht (Germany); Weinberg, Annelie [Department of Orthopedics and Orthopedic Surgery, Medical University of Graz (Austria); Luthringer, Bérengère J.C. [Helmholtz-Zentrum Geesthacht, Institute of Material Research, Department for Structural Research on Macromolecules, Geesthacht (Germany)

    2017-03-01

    Biodegradable magnesium (Mg)-based materials are a potential alternative to permanent implants for application in children. Nevertheless effects of those materials on growth plate cartilage and chondrogenesis have not been previously evaluated. In vitro differentiation of ATDC5 cells was evaluated under the influence of pure Mg (PMg), Mg with 10 wt% of gadolinium (Mg-10Gd) and Mg with 2 wt% of silver (Mg-2Ag) degradation products (extracts) and direct cell culture on the materials. Gene expression showed an inhibitory effect on ATDC5 mineralization with the three extracts and a chondrogenic potential of Mg-10Gd. Cells cultured in Mg-10Gd and Mg-2Ag extracts showed the same proliferation and morphology than cells cultured in growth conditions. Mg-10Gd induced an increase in production of ECM and a bigger cell size, similar to the effects found with differentiation conditions. An increased metabolic activity was observed in cells cultured under the influence of Mg-10Gd extracts, indicated by an acidic pH during most of the culture period. After 7 days of culture on the materials, ATDC5 growth, distribution and ECM synthesis were higher on Mg-10Gd samples, followed by Mg-2Ag and PMg, which was influenced by the homogeneity and composition of the degradation layer. This study confirmed the tolerance of ATDC5 cells to Mg-based materials and a chondrogenic effect of Mg-10Gd. Further studies in vitro and in vivo are necessary to evaluate cell reactions to those materials, as well as the effects on bone growth and the biocompatibility of the alloying system in the body. - Highlights: • Degradation of PMg, and Mg-2Ag do not influence ATDC5 cells growth and chondrogenic redifferentiation. • Mg-10Gd enhances fast chondrogenic redifferentiation and expression of hyperthrophic markers on ATDC5 cells. • Further evaluation of the effects of PMg, Mg-10Gd and Mg-2Ag in vivo are necessary to confirm its potential for application in growing bones.

  8. Chondrogenic differentiation of ATDC5-cells under the influence of Mg and Mg alloy degradation

    International Nuclear Information System (INIS)

    Martinez Sanchez, Adela H.; Feyerabend, Frank; Laipple, Daniel; Willumeit-Römer, Regine; Weinberg, Annelie; Luthringer, Bérengère J.C.

    2017-01-01

    Biodegradable magnesium (Mg)-based materials are a potential alternative to permanent implants for application in children. Nevertheless effects of those materials on growth plate cartilage and chondrogenesis have not been previously evaluated. In vitro differentiation of ATDC5 cells was evaluated under the influence of pure Mg (PMg), Mg with 10 wt% of gadolinium (Mg-10Gd) and Mg with 2 wt% of silver (Mg-2Ag) degradation products (extracts) and direct cell culture on the materials. Gene expression showed an inhibitory effect on ATDC5 mineralization with the three extracts and a chondrogenic potential of Mg-10Gd. Cells cultured in Mg-10Gd and Mg-2Ag extracts showed the same proliferation and morphology than cells cultured in growth conditions. Mg-10Gd induced an increase in production of ECM and a bigger cell size, similar to the effects found with differentiation conditions. An increased metabolic activity was observed in cells cultured under the influence of Mg-10Gd extracts, indicated by an acidic pH during most of the culture period. After 7 days of culture on the materials, ATDC5 growth, distribution and ECM synthesis were higher on Mg-10Gd samples, followed by Mg-2Ag and PMg, which was influenced by the homogeneity and composition of the degradation layer. This study confirmed the tolerance of ATDC5 cells to Mg-based materials and a chondrogenic effect of Mg-10Gd. Further studies in vitro and in vivo are necessary to evaluate cell reactions to those materials, as well as the effects on bone growth and the biocompatibility of the alloying system in the body. - Highlights: • Degradation of PMg, and Mg-2Ag do not influence ATDC5 cells growth and chondrogenic redifferentiation. • Mg-10Gd enhances fast chondrogenic redifferentiation and expression of hyperthrophic markers on ATDC5 cells. • Further evaluation of the effects of PMg, Mg-10Gd and Mg-2Ag in vivo are necessary to confirm its potential for application in growing bones.

  9. Gelatin-Based Hydrogels Promote Chondrogenic Differentiation of Human Adipose Tissue-Derived Mesenchymal Stem Cells In Vitro

    Directory of Open Access Journals (Sweden)

    Achim Salamon

    2014-02-01

    Full Text Available Due to the weak regeneration potential of cartilage, there is a high clinical incidence of articular joint disease, leading to a strong demand for cartilaginous tissue surrogates. The aim of this study was to evaluate a gelatin-based hydrogel for its suitability to support chondrogenic differentiation of human mesenchymal stem cells. Gelatin-based hydrogels are biodegradable, show high biocompatibility, and offer possibilities to introduce functional groups and/or ligands. In order to prove their chondrogenesis-supporting potential, a hydrogel film was developed and compared with standard cell culture polystyrene regarding the differentiation behavior of human mesenchymal stem cells. Cellular basis for this study were human adipose tissue-derived mesenchymal stem cells, which exhibit differentiation potential along the adipogenic, osteogenic and chondrogenic lineage. The results obtained show a promotive effect of gelatin-based hydrogels on chondrogenic differentiation of mesenchymal stem cells in vitro and therefore encourage subsequent in vivo studies.

  10. Gelatin-Based Hydrogels Promote Chondrogenic Differentiation of Human Adipose Tissue-Derived Mesenchymal Stem Cells In Vitro

    Science.gov (United States)

    Salamon, Achim; van Vlierberghe, Sandra; van Nieuwenhove, Ine; Baudisch, Frank; Graulus, Geert-Jan; Benecke, Verena; Alberti, Kristin; Neumann, Hans-Georg; Rychly, Joachim; Martins, José C.; Dubruel, Peter; Peters, Kirsten

    2014-01-01

    Due to the weak regeneration potential of cartilage, there is a high clinical incidence of articular joint disease, leading to a strong demand for cartilaginous tissue surrogates. The aim of this study was to evaluate a gelatin-based hydrogel for its suitability to support chondrogenic differentiation of human mesenchymal stem cells. Gelatin-based hydrogels are biodegradable, show high biocompatibility, and offer possibilities to introduce functional groups and/or ligands. In order to prove their chondrogenesis-supporting potential, a hydrogel film was developed and compared with standard cell culture polystyrene regarding the differentiation behavior of human mesenchymal stem cells. Cellular basis for this study were human adipose tissue-derived mesenchymal stem cells, which exhibit differentiation potential along the adipogenic, osteogenic and chondrogenic lineage. The results obtained show a promotive effect of gelatin-based hydrogels on chondrogenic differentiation of mesenchymal stem cells in vitro and therefore encourage subsequent in vivo studies. PMID:28788517

  11. Osteochondral defect repair using bilayered hydrogels encapsulating both chondrogenically and osteogenically pre-differentiated mesenchymal stem cells in a rabbit model

    NARCIS (Netherlands)

    Lam, J.; Lu, S.; Lee, E.J.; Trachtenberg, J.E.; Meretoja, V.V.; Dahlin, R.L.; van den Beucken, J.J.; Tabata, Y.; Wong, M.E.; Jansen, J.A.; Mikos, A.G.; Kasper, F.K.

    2014-01-01

    OBJECTIVE: To investigate the ability of cell-laden bilayered hydrogels encapsulating chondrogenically and osteogenically (OS) pre-differentiated mesenchymal stem cells (MSCs) to effect osteochondral defect repair in a rabbit model. By varying the period of chondrogenic pre-differentiation from 7

  12. Serum estradiol levels associated with specific gene expression patterns in normal breast tissue and in breast carcinomas

    International Nuclear Information System (INIS)

    Haakensen, Vilde D; Børresen-Dale, Anne-Lise; Helland, Åslaug; Bjøro, Trine; Lüders, Torben; Riis, Margit; Bukholm, Ida K; Kristensen, Vessela N; Troester, Melissa A; Homen, Marit M; Ursin, Giske

    2011-01-01

    High serum levels of estradiol are associated with increased risk of postmenopausal breast cancer. Little is known about the gene expression in normal breast tissue in relation to levels of circulating serum estradiol. We compared whole genome expression data of breast tissue samples with serum hormone levels using data from 79 healthy women and 64 breast cancer patients. Significance analysis of microarrays (SAM) was used to identify differentially expressed genes and multivariate linear regression was used to identify independent associations. Six genes (SCGB3A1, RSPO1, TLN2, SLITRK4, DCLK1, PTGS1) were found differentially expressed according to serum estradiol levels (FDR = 0). Three of these independently predicted estradiol levels in a multivariate model, as SCGB3A1 (HIN1) and TLN2 were up-regulated and PTGS1 (COX1) was down-regulated in breast samples from women with high serum estradiol. Serum estradiol, but none of the differentially expressed genes were significantly associated with mammographic density, another strong breast cancer risk factor. In breast carcinomas, expression of GREB1 and AREG was associated with serum estradiol in all cancers and in the subgroup of estrogen receptor positive cases. We have identified genes associated with serum estradiol levels in normal breast tissue and in breast carcinomas. SCGB3A1 is a suggested tumor suppressor gene that inhibits cell growth and invasion and is methylated and down-regulated in many epithelial cancers. Our findings indicate this gene as an important inhibitor of breast cell proliferation in healthy women with high estradiol levels. In the breast, this gene is expressed in luminal cells only and is methylated in non-BRCA-related breast cancers. The possibility of a carcinogenic contribution of silencing of this gene for luminal, but not basal-like cancers should be further explored. PTGS1 induces prostaglandin E2 (PGE2) production which in turn stimulates aromatase expression and hence increases the

  13. Impact of human platelet lysate on the expansion and chondrogenic capacity of cultured human chondrocytes for cartilage cell therapy.

    Science.gov (United States)

    Sykes, J G; Kuiper, J H; Richardson, J B; Roberts, S; Wright, K T; Kuiper, N J

    2018-05-01

    High hopes have been pinned on regenerative medicine strategies in order to prevent the progression of cartilage damage to osteoarthritis, particularly by autologous chondrocyte implantation (ACI). The loss of chondrocyte phenotype during in vitro monolayer expansion, a necessary step to obtain sufficient cell numbers, may be a key limitation in ACI. In this study, it was determined whether a shorter monolayer expansion approach could improve chondrogenic differentiation. The effects of two supplement types, foetal bovine serum (FBS) and Stemulate™ (a commercial source of human platelet lysate), on the expansion and re-differentiation potential of human chondrocytes, isolated from five individuals, were compared. Chondrocytes were expanded with 10 % FBS or 10 % Stemulate™. Pellets were cultured for 28 d in chondrogenic differentiation medium and assessed for the presence of cartilage matrix molecules and genes associated with chondrogenicity. Stemulate™ significantly enhanced the proliferation rate [average population doubling times: FBS, 25.07 ± 6.98 d (standard error of the mean, SEM) vs. Stemulate™, 13.10 ± 2.57 d (SEM)]. Sulphated glycosaminoglycans (sGAG), total collagen and qRT-PCR analyses of cartilage genes showed that FBS-expanded chondrocytes demonstrated significantly better chondrogenic capacity than Stemulate™-expanded chondrocytes. Histologically, FBS-expanded chondrocyte pellets appeared to be more stable, with a more intense staining for toluidine blue, indicating a greater chondrogenic capacity. Although Stemulate™ positively influenced chondrocyte proliferation, it had a negative effect on chondrogenic differentiation potential. This suggested that, in the treatment of cartilage defects, Stemulate™ might not be the ideal supplement for expanding chondrocytes (which maintained a chondrocyte phenotype) and, hence, for cell therapies (including ACI).

  14. Impact of human platelet lysate on the expansion and chondrogenic capacity of cultured human chondrocytes for cartilage cell therapy

    Directory of Open Access Journals (Sweden)

    JG Sykes

    2018-05-01

    Full Text Available High hopes have been pinned on regenerative medicine strategies in order to prevent the progression of cartilage damage to osteoarthritis, particularly by autologous chondrocyte implantation (ACI. The loss of chondrocyte phenotype during in vitro monolayer expansion, a necessary step to obtain sufficient cell numbers, may be a key limitation in ACI. In this study, it was determined whether a shorter monolayer expansion approach could improve chondrogenic differentiation. The effects of two supplement types, foetal bovine serum (FBS and Stemulate™ (a commercial source of human platelet lysate, on the expansion and re-differentiation potential of human chondrocytes, isolated from five individuals, were compared. Chondrocytes were expanded with 10 % FBS or 10 % Stemulate™. Pellets were cultured for 28 d in chondrogenic differentiation medium and assessed for the presence of cartilage matrix molecules and genes associated with chondrogenicity. Stemulate™ significantly enhanced the proliferation rate [average population doubling times: FBS, 25.07 ± 6.98 d (standard error of the mean, SEM vs. Stemulate™, 13.10 ± 2.57 d (SEM]. Sulphated glycosaminoglycans (sGAG, total collagen and qRT-PCR analyses of cartilage genes showed that FBS-expanded chondrocytes demonstrated significantly better chondrogenic capacity than Stemulate™-expanded chondrocytes. Histologically, FBS-expanded chondrocyte pellets appeared to be more stable, with a more intense staining for toluidine blue, indicating a greater chondrogenic capacity. Although Stemulate™ positively influenced chondrocyte proliferation, it had a negative effect on chondrogenic differentiation potential. This suggested that, in the treatment of cartilage defects, Stemulate™ might not be the ideal supplement for expanding chondrocytes (which maintained a chondrocyte phenotype and, hence, for cell therapies (including ACI.

  15. A reliable protocol for the isolation of viable, chondrogenically differentiated human mesenchymal stem cells from high-density pellet cultures.

    Science.gov (United States)

    Ullah, Mujib; Hamouda, Houda; Stich, Stefan; Sittinger, Michael; Ringe, Jochen

    2012-12-01

    Administration of chondrogenically differentiated mesenchymal stem cells (MSC) is discussed as a promising approach for the regenerative treatment of injured or diseased cartilage. The high-density pellet culture is the standard culture for chondrogenic differentiation, but cells in pellets secrete extracellular matrix (ECM) that they become entrapped in. Protocols for cell isolation from pellets often result in cell damage and dedifferentiation towards less differentiated MSC. Therefore, our aim was to develop a reliable protocol for the isolation of viable, chondrogenically differentiated MSC from high-density pellet cultures. Human bone marrow MSC were chondrogenically stimulated with transforming growth factor-β3, and the cartilaginous structure of the pellets was verified by alcian blue staining of cartilage proteoglycans, antibody staining of cartilage collagen type II, and quantitative real-time reverse-transcription polymerase chain reaction of the marker genes COL2A1 and SOX9. Trypsin and collagenases II and P were tested alone or in combination, and for different concentrations and times, to find a protocol for optimized pellet digestion. Whereas trypsin was not able to release viable cells, 90-min digestion with 300 U of collagenase II, 20 U of collagenase P, and 2 mM CaCl2 worked quite well and resulted in about 2.5×10(5) cells/pellet. The protocol was further optimized for the separation of released cells and ECM from each other. Cells were alcian blue and collagen type II positive and expressed COL2A1 and SOX9, verifying a chondrogenic character. However, they had different morphological shapes. The ECM was also uniformly alcian blue and collagen type II positive but showed different organizational and structural forms. To conclude, our protocol allows the reliable isolation of a defined number of viable, chondrogenically differentiated MSC from high-density pellet cultures. Such cells, as well as the ECM components, are of interest as

  16. Comparative study of the chondrogenic potential of human bone marrow stromal cells, neonatal chondrocytes and adult chondrocytes

    International Nuclear Information System (INIS)

    Saha, Sushmita; Kirkham, Jennifer; Wood, David; Curran, Stephen; Yang, Xuebin

    2010-01-01

    Research highlights: → This study has characterised three different cell types under conditions similar to those used for autologous chondrocyte implantation (ACI) for applications in cartilage repair/regeneration. → Compared for the first time the chondrogenic potential of neonatal chondrocytes with human bone marrow stromal cells (HBMSCs) and adult chondrocytes. → Demonstrated that adult chondrocytes hold greatest potential for use in ACI based on their higher proliferation rates, lower alkaline phosphatise activity and enhanced expression of chondrogenic genes. → Demonstrated the need for chondroinduction as a necessary pre-requisite to efficient chondrogenesis in vitro and, by extrapolation, for cell based therapy (e.g. ACI or cartilage tissue engineering). -- Abstract: Cartilage tissue engineering is still a major clinical challenge with optimisation of a suitable source of cells for cartilage repair/regeneration not yet fully addressed. The aims of this study were to compare and contrast the differences in chondrogenic behaviour between human bone marrow stromal cells (HBMSCs), human neonatal and adult chondrocytes to further our understanding of chondroinduction relative to cell maturity and to identify factors that promote chondrogenesis and maintain functional homoeostasis. Cells were cultured in monolayer in either chondrogenic or basal medium, recapitulating procedures used in existing clinical procedures for cell-based therapies. Cell doubling time, morphology and alkaline phosphatase specific activity (ALPSA) were determined at different time points. Expression of chondrogenic markers (SOX9, ACAN and COL2A1) was compared via real time polymerase chain reaction. Amongst the three cell types studied, HBMSCs had the highest ALPSA in basal culture and lowest ALPSA in chondrogenic media. Neonatal chondrocytes were the most proliferative and adult chondrocytes had the lowest ALPSA in basal media. Gene expression analysis revealed a difference in the

  17. Comparative study of the chondrogenic potential of human bone marrow stromal cells, neonatal chondrocytes and adult chondrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Saha, Sushmita [Biomaterials and Tissue Engineering Group, Leeds Dental Institute, University of Leeds, LS29LU (United Kingdom); Kirkham, Jennifer [Biomineralisation Group, Leeds Dental Institute, University of Leeds, LS29LU (United Kingdom); NIHR Leeds Musculoskeletal Biomedical Research Unit, University of Leeds, Chapel Allerton Hospital, Leeds LS74SA (United Kingdom); Wood, David [Biomaterials and Tissue Engineering Group, Leeds Dental Institute, University of Leeds, LS29LU (United Kingdom); Curran, Stephen [Smith and Nephew Research Centre, YO105DF (United Kingdom); Yang, Xuebin, E-mail: X.B.Yang@leeds.ac.uk [Biomaterials and Tissue Engineering Group, Leeds Dental Institute, University of Leeds, LS29LU (United Kingdom); NIHR Leeds Musculoskeletal Biomedical Research Unit, University of Leeds, Chapel Allerton Hospital, Leeds LS74SA (United Kingdom)

    2010-10-22

    Research highlights: {yields} This study has characterised three different cell types under conditions similar to those used for autologous chondrocyte implantation (ACI) for applications in cartilage repair/regeneration. {yields} Compared for the first time the chondrogenic potential of neonatal chondrocytes with human bone marrow stromal cells (HBMSCs) and adult chondrocytes. {yields} Demonstrated that adult chondrocytes hold greatest potential for use in ACI based on their higher proliferation rates, lower alkaline phosphatise activity and enhanced expression of chondrogenic genes. {yields} Demonstrated the need for chondroinduction as a necessary pre-requisite to efficient chondrogenesis in vitro and, by extrapolation, for cell based therapy (e.g. ACI or cartilage tissue engineering). -- Abstract: Cartilage tissue engineering is still a major clinical challenge with optimisation of a suitable source of cells for cartilage repair/regeneration not yet fully addressed. The aims of this study were to compare and contrast the differences in chondrogenic behaviour between human bone marrow stromal cells (HBMSCs), human neonatal and adult chondrocytes to further our understanding of chondroinduction relative to cell maturity and to identify factors that promote chondrogenesis and maintain functional homoeostasis. Cells were cultured in monolayer in either chondrogenic or basal medium, recapitulating procedures used in existing clinical procedures for cell-based therapies. Cell doubling time, morphology and alkaline phosphatase specific activity (ALPSA) were determined at different time points. Expression of chondrogenic markers (SOX9, ACAN and COL2A1) was compared via real time polymerase chain reaction. Amongst the three cell types studied, HBMSCs had the highest ALPSA in basal culture and lowest ALPSA in chondrogenic media. Neonatal chondrocytes were the most proliferative and adult chondrocytes had the lowest ALPSA in basal media. Gene expression analysis revealed

  18. Circulating Estradiol Regulates Brain-Derived Estradiol via Actions at GnRH Receptors to Impact Memory in Ovariectomized Rats.

    Science.gov (United States)

    Nelson, Britta S; Black, Katelyn L; Daniel, Jill M

    2016-01-01

    Systemic estradiol treatment enhances hippocampus-dependent memory in ovariectomized rats. Although these enhancements are traditionally thought to be due to circulating estradiol, recent data suggest these changes are brought on by hippocampus-derived estradiol, the synthesis of which depends on gonadotropin-releasing hormone (GnRH) activity. The goal of the current work is to test the hypothesis that peripheral estradiol affects hippocampus-dependent memory through brain-derived estradiol regulated via hippocampal GnRH receptor activity. In the first experiment, intracerebroventricular infusion of letrozole, which prevents the synthesis of estradiol, blocked the ability of peripheral estradiol administration in ovariectomized rats to enhance hippocampus-dependent memory in a radial-maze task. In the second experiment, hippocampal infusion of antide, a long-lasting GnRH receptor antagonist, blocked the ability of peripheral estradiol administration in ovariectomized rats to enhance hippocampus-dependent memory. In the third experiment, hippocampal infusion of GnRH enhanced hippocampus-dependent memory, the effects of which were blocked by letrozole infusion. Results indicate that peripheral estradiol-induced enhancement of cognition is mediated by brain-derived estradiol via hippocampal GnRH receptor activity.

  19. Chondrogenic differentiation of mesenchymal stem cells in a leakproof collagen sponge

    International Nuclear Information System (INIS)

    Chen Guoping; Akahane, Daisuke; Kawazoe, Naoki; Yamamoto, Katsuyuki; Tateishi, Tetsuya

    2008-01-01

    A three-dimensional culture of mesenchymal stem cells (MSCs) in a porous scaffold has been developed as a promising strategy for cartilage tissue engineering. The chondrogenic differentiation of MSCs derived from human bone marrow was studied by culturing the cells in a novel scaffold constructed of leakproof collagen sponge. All the surfaces of the collagen sponge except the top were wrapped with a membrane that has pores smaller than the cells to protect against cell leakage during cell seeding. The cells adhered to the collagen, distributed evenly, and proliferated to fill the spaces in the sponge. Cell seeding efficiency was greater than 95%. The MSCs cultured in the collagen sponge in the presence of TGF-β3 and BMP6 expressed a high level of genes encoding type II and type X collagen, sox9, and aggrecan. Histological examination by HE staining indicated that the differentiated cells showed a round morphology. The extracellular matrices were positively stained by safranin O and toluidine blue. Immunostaining with anti-type II collagen and anti-cartilage proteoglycan showed that type II collagen and cartilage proteoglycan were detected around the cells. These results suggest the chondrogenic differentiation of MSCs when cultured in the collagen sponge in the presence of TGF-β3 and BMP6

  20. Evaluation of insulin medium or chondrogenic medium on proliferation and chondrogenesis of ATDC5 cells.

    Science.gov (United States)

    Yao, Yongchang; Zhai, Zhichen; Wang, Yingjun

    2014-01-01

    The ATDC5 cell line is regarded as an excellent cell model for chondrogenesis. In most studies with ATDC5 cells, insulin medium (IM) was used to induce chondrogenesis while chondrogenic medium (CM), which was usually applied in chondrogenesis of mesenchymal stem cells (MSCs), was rarely used for ATDC5 cells. This study was mainly designed to investigate the effect of IM, CM, and growth medium (GM) on chondrogenesis of ATDC5 cells. ATDC5 cells were, respectively, cultured in IM, CM, and GM for a certain time. Then the proliferation and the chondrogenesis progress of cells in these groups were analyzed. Compared with CM and GM, IM promoted the proliferation of cells significantly. CM was effective for enhancement of cartilage specific markers, while IM induced the cells to express endochondral ossification related genes. Although GAG deposition per cell in CM group was significantly higher than that in IM and GM groups, the total GAG contents in IM group were the most. This study demonstrated that CM focused on induction of chondrogenic differentiation while IM was in favor of promoting proliferation and expression of endochondral ossification related genes. Combinational use of these two media would be more beneficial to bone/cartilage repair.

  1. Different Chondrogenic Potential among Human Induced Pluripotent Stem Cells from Diverse Origin Primary Cells

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    Yeri Alice Rim

    2018-01-01

    Full Text Available Scientists have tried to reprogram various origins of primary cells into human induced pluripotent stem cells (hiPSCs. Every somatic cell can theoretically become a hiPSC and give rise to targeted cells of the human body. However, there have been debates on the controversy about the differentiation propensity according to the origin of primary cells. We reprogrammed hiPSCs from four different types of primary cells such as dermal fibroblasts (DF, n=3, peripheral blood mononuclear cells (PBMC, n=3, cord blood mononuclear cells (CBMC, n=3, and osteoarthritis fibroblast-like synoviocytes (OAFLS, n=3. Established hiPSCs were differentiated into chondrogenic pellets. All told, cartilage-specific markers tended to express more by the order of CBMC > DF > PBMC > FLS. Origin of primary cells may influence the reprogramming and differentiation thereafter. In the context of chondrogenic propensity, CBMC-derived hiPSCs can be a fairly good candidate cell source for cartilage regeneration. The differentiation of hiPSCs into chondrocytes may help develop “cartilage in a dish” in the future. Also, the ideal cell source of hiPSC for chondrogenesis may contribute to future application as well.

  2. Evaluation of Insulin Medium or Chondrogenic Medium on Proliferation and Chondrogenesis of ATDC5 Cells

    Directory of Open Access Journals (Sweden)

    Yongchang Yao

    2014-01-01

    Full Text Available Background. The ATDC5 cell line is regarded as an excellent cell model for chondrogenesis. In most studies with ATDC5 cells, insulin medium (IM was used to induce chondrogenesis while chondrogenic medium (CM, which was usually applied in chondrogenesis of mesenchymal stem cells (MSCs, was rarely used for ATDC5 cells. This study was mainly designed to investigate the effect of IM, CM, and growth medium (GM on chondrogenesis of ATDC5 cells. Methods. ATDC5 cells were, respectively, cultured in IM, CM, and GM for a certain time. Then the proliferation and the chondrogenesis progress of cells in these groups were analyzed. Results. Compared with CM and GM, IM promoted the proliferation of cells significantly. CM was effective for enhancement of cartilage specific markers, while IM induced the cells to express endochondral ossification related genes. Although GAG deposition per cell in CM group was significantly higher than that in IM and GM groups, the total GAG contents in IM group were the most. Conclusion. This study demonstrated that CM focused on induction of chondrogenic differentiation while IM was in favor of promoting proliferation and expression of endochondral ossification related genes. Combinational use of these two media would be more beneficial to bone/cartilage repair.

  3. Periodic heat shock accelerated the chondrogenic differentiation of human mesenchymal stem cells in pellet culture.

    Directory of Open Access Journals (Sweden)

    Jing Chen

    Full Text Available Osteoarthritis (OA is one of diseases that seriously affect elderly people's quality of life. Human mesenchymal stem cells (hMSCs offer a potential promise for the joint repair in OA patients. However, chondrogenic differentiation from hMSCs in vitro takes a long time (∼ 6 weeks and differentiated cells are still not as functionally mature as primary isolated chondrocytes, though chemical stimulations and mechanical loading have been intensively studied to enhance the hMSC differentiation. On the other hand, thermal stimulations of hMSC chondrogenesis have not been well explored. In this study, the direct effects of mild heat shock (HS on the differentiation of hMSCs into chondrocytes in 3D pellet culture were investigated. Periodic HS at 41 °C for 1 hr significantly increased sulfated glycosaminoglycan in 3D pellet culture at Day 10 of chondrogenesis. Immunohistochemical and Western Blot analyses revealed an increased expression of collagen type II and aggrecan in heat-shocked pellets than non heat-shocked pellets on Day 17 of chondrogenesis. In addition, HS also upregulated the expression of collagen type I and X as well as heat shock protein 70 on Day 17 and 24 of differentiation. These results demonstrate that HS accelerated the chondrogenic differentiation of hMSCs and induced an early maturation of chondrocytes differentiated from hMSCs. The results of this study will guide the design of future protocols using thermal treatments to facilitate cartilage regeneration with human mesenchymal stem cells.

  4. Long-Term Expandable SOX9+ Chondrogenic Ectomesenchymal Cells from Human Pluripotent Stem Cells

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    Katsutsugu Umeda

    2015-04-01

    Full Text Available Here we report the successful generation and long-term expansion of SOX9-expressing CD271+PDGFRα+CD73+ chondrogenic ectomesenchymal cells from the PAX3/SOX10/FOXD3-expressing MIXL1−CD271hiPDGFRαloCD73− neural crest-like progeny of human pluripotent stem cells in a chemically defined medium supplemented with Nodal/Activin/transforming growth factorβ (TGFβ inhibitor and fibroblast growth factor (FGF. When “primed” with TGFβ, such cells efficiently formed translucent cartilage particles, which were completely mineralized in 12 weeks in immunocompromized mice. The ectomesenchymal cells were expandable without loss of chondrogenic potential for at least 16 passages. They maintained normal karyotype for at least 10 passages and expressed genes representing embryonic progenitors (SOX4/12, LIN28A/B, cranial mesenchyme (ALX1/3/4, and chondroprogenitors (SOX9, COL2A1 of neural crest origin (SOX8/9, NGFR, NES. Ectomesenchyme is a source of many craniofacial bone and cartilage structures. The method we describe for obtaining a large quantity of human ectomesenchymal cells will help to model craniofacial disorders in vitro and potentially provide cells for the repair of craniofacial damage.

  5. Growth factor combination for chondrogenic induction from human mesenchymal stem cell

    International Nuclear Information System (INIS)

    Indrawattana, Nitaya; Chen Guoping; Tadokoro, Mika; Shann, Linzi H.; Ohgushi, Hajime; Tateishi, Tetsuya; Tanaka, Junzo; Bunyaratvej, Ahnond

    2004-01-01

    During the last decade, many strategies for cartilage engineering have been emerging. Stem cell induction is one of the possible approaches for cartilage engineering. The mesenchymal stem cells (MSCs) with their pluripotency and availability have been demonstrated to be an attractive cell source. It needs the stimulation with cell growth factors to make the multipluripotent MSCs differentiate into chondrogenic lineage. We have shown particular patterns of in vitro chondrogenesis induction on human bone marrow MSCs (hBMSCs) by cycling the growth factors. The pellet cultures of hBMSCs were prepared for chondrogenic induction. Growth factors: TGF-β3, BMP-6, and IGF-1 were used in combination for cell induction. Gene expression, histology, immunohistology, and real-time PCR methods were measured on days 21 after cell induction. As shown by histology and immunohistology, the induced cells have shown the feature of chondrocytes in their morphology and extracellular matrix in both inducing patterns of combination and cycling induction. Moreover, the real-time PCR assay has shown the expression of gene markers of chondrogenesis, collagen type II and aggrecan. This study has demonstrated that cartilage tissue can be created from bone marrow mesenchymal stem cells. Interestingly, the combined growth factors TGF-β3 and BMP-6 or TGF-β3 and IGF-1 were more effective for chondrogenesis induction as shown by the real-time PCR assay. The combination of these growth factors may be the important key for in vitro chondrogenesis induction

  6. Chondrogenic differentiation of human mesenchymal stem cells cultured in a cobweb-like biodegradable scaffold

    International Nuclear Information System (INIS)

    Chen Guoping; Liu Dechang; Tadokoro, Mika; Hirochika, Rei; Ohgushi, Hajime; Tanaka, Junzo; Tateishi, Tetsuya

    2004-01-01

    Human mesenchymal stem cells (MSCs) were cultured in vitro in a cobweb-like biodegradable polymer scaffold: a poly(DL-lactic-co-glycolic acid)-collagen hybrid mesh in serum-free DMEM containing TGF-β3 for 1-10 weeks. The cells adhered to the hybrid mesh, distributed evenly, and proliferated to fill the spaces in the scaffold. The ability of the cells to express gene encoding type I collagen decreased, whereas its ability to express type II collagen and aggrecan increased. Histological examination by HE staining indicated that the cells showed fibroblast morphology at the early stage and became round after culture for 4 weeks. The cartilaginous matrices were positively stained by safranin O and toluidine blue. Immunostaining with anti-type II collagen and anti-cartilage proteoglycan showed that type II collagen and cartilage proteoglycan were detected around the cells. In addition, a homogeneous distribution of cartilaginous extracellular matrices was detected around the cells. These results suggest the chondrogenic differentiation of the mesenchymal stem cells in the hybrid mesh. The PLGA-collagen hybrid mesh enabled the aggregation of mesenchymal stem cells and provided a promotive microenvironment for the chondrogenic differentiation of the MSCs

  7. The chondrogenic response to exercise in the proximal femur of normal and mdx mice

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    Nye David J

    2010-09-01

    Full Text Available Abstract Background Submaximal exercise is used in the management of muscular dystrophy. The effects of mechanical stimulation on skeletal development are well understood, although its effects on cartilage growth have yet to be investigated in the dystrophic condition. The objective of this study was to investigate the chondrogenic response to voluntary exercise in dystrophin-deficient mice. Methods Control and dystrophin-deficient (mdx mice were divided into sedentary and exercise-treated groups and tested for chondral histomorphometric differences at the proximal femur. Results Control mice ran 7 km/week further than mdx mice on average, but this difference was not statistically significant (P > 0.05. However, exercised control mice exhibited significantly enlarged femur head diameter, articular cartilage thickness, articular cartilage tissue area, and area of calcified cartilage relative to sedentary controls and exercised mdx mice (P Conclusions Mdx mice exhibit a reduced chondrogenic response to increased mechanical stimulation relative to controls. However, no significant reduction in articular dimensions was found, indicating loss of chondral tissue may not be a clinical concern with dystrophinopathy.

  8. PI3K/Akt Activated by GPR30 and Src Regulates 17β-Estradiol-Induced Cultured Immature Boar Sertoli Cells Proliferation.

    Science.gov (United States)

    Yang, Wei-Rong; Zhu, Feng-Wei; Zhang, Jiao-Jiao; Wang, Yi; Zhang, Jia-Hua; Lu, Cheng; Wang, Xian-Zhong

    2016-05-24

    Sertoli cell (SC) is a key element in the process of spermatogenesis. Accumulating research show that estrogen plays an important role in regulating boar SC proliferation. However, it is unclear whether phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B (PI3K/Akt) is involved in this process. In the present study, the role of PI3K/Akt on the 17β-estradiol-induced piglet SC proliferation was explored. In addition, we also explained the roles of G-protein-coupled estrogen receptor (GPR30) and Sarcoma protein (Src) in this process. Our study demonstrated that, 17β-estradiol induced activation of PI3K in a time-dependent manner. Both G-15 (an antagonist of GPR30, 0.1 μmol/L) and PP2 (an inhibitor of Src, 2.0 μmol/L) inhibited 17β-estradiol-induced activation of PI3K, reduced SC proliferation, and decreased messenger RNA (mRNA) and protein expression of S-phase kinase-associated protein 2 (Skp2). We also found that 17β-estradiol induced activation of Akt in a time-dependent manner. Both LY294002 (an inhibitor of PI3K) and 10-DEBC (an inhibitor of Akt) significantly reduced 17β-estradiol-induced SC proliferation and reduced mRNA and protein expression of Skp2. In addition, LY294002 inhibited 17β-estradiol-induced activation of Akt. The results indicated that 17β-estradiol regulates SC proliferation by activating PI3K/Akt. Both GPR30 and Src are involved in 17β-estradiol-induced phosphorylation of PI3K/Akt. Activation of PI3K/Akt enhances the expression of Skp2, which promotes SC proliferation. © The Author(s) 2016.

  9. 17β-estradiol regulates the differentiation of cementoblasts via Notch signaling cascade

    Energy Technology Data Exchange (ETDEWEB)

    Liao, Jing; Zhou, Zeyuan; Huang, Li; Li, Yuyu [Department of Orthodontics, The State Key Laboratory of Oral Disease, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan Province (China); Li, Jingtao [Department of Oral and Maxillofacial Surgery, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan Province (China); Zou, Shujuan, E-mail: drzsj@scu.edu.cn [Department of Orthodontics, The State Key Laboratory of Oral Disease, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan Province (China)

    2016-08-12

    Estrogen has been well recognized as a key factor in the homeostasis of bone and periodontal tissue, but the way it regulates the activities of cementoblasts, the cell population maintaining cementum has not been fully understood. In this study, we examined the expression of estrogen receptor in OCCM-30 cells and the effect of 17β-estradiol (E2) on the proliferation and differentiation of OCCM-30 cells. We found that both estrogen receptor α and β were expressed in OCCM-30 cells. E2 exerted no significant influence on the proliferation of OCCM-30 cells, but inhibited the transcription and translation of BSP and Runx2 in the early phase of osteogenic induction except the BSP mRNA. Afterwards in the late phase of osteogenic induction, E2 enhanced the transcription and translation of BSP and Runx2 and promoted the calcium deposition. In addition, the expression level of Notch1, NICD and Hey1 mRNAs responded to exogenous E2 in a pattern similar to that of the osteoblastic markers. DAPT could attenuate the effect of E2 on the expression of osteoblastic markers. These findings indicated that E2 might regulate the differentiation of cementoblasts via Notch signaling. - Highlights: • 17β-estradiol showed no significant effect on the proliferation of cementoblasts. • 17β-estradiol promoted the osteoblastic differentiation of cementoblasts despite of an early transient inhibition. • Notch signaling was regulated by 17β-estradiol and was responsible for mediating the effect of E2 on cementoblasts. • Hey1 might display an opposite expression pattern to Notch signaling in certain circumstances.

  10. Effect of Estradiol-17β Injection on Gonad Development of White Shrimp (Litopenaeus vannamei

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    . Tarsim

    2007-01-01

    Full Text Available The methods for hormonal control of shrimp reproduction are very limited, and only eyestalk ablation is used to induce ovarian development and spawning in shrimp farming. The occurrence of vertebrate-type steroid hormones in crustaceans have been reported, however, their physiological role are not sufficiently understood. The present study analyzed the effect of estradiol-17β injection on gonad development of white shrimp, Litopenaeus vannamei. The estradiol-17β dose 0.10 μg/g body weight were used. The treatments consisted of control, single injection (day 0 and double injection (day 0 and 6. The females broodstock were cultured for 12 days. The result showed that estradiol-17β had positive effect on gonad development. The gonado somatic index (GSI and oocytes diameter in treatment larger than the control. Double injection had highest effect with ∆GSI and oocytes diameter was 0.453  and 23.97 µm, respectively. The only oocytes previtelogenesis was found in gonad. It indicated that estradiol-17β important to induce endogenous vitellogenesis. Gonad development probably affected by gonad inhibiting hormone in the eyestalk. It was inhibited oocyte maturation. The polypeptide sub unit was observed in vitellin of ovari by SDS-PAGE. The molecular weights of approximately 95, 98, 109 and two units higher than 118 kDa of protein marker. Keywords: Gonad, estradiol-17β, oocyte, Litopenaeus vannamei   ABSTRAK Teknologi reproduksi dalam pembenihan udang belum mengalami perkembangan yang signifikan.  Pada umumnya untuk mempercepat kematangan gonad induk udang digunakan teknik ablasi. Mekanisme dan peranan hormon pada proses reproduksi udang belum banyak diketahui. Keberadaan hormon steroid pada krustase telah dikemukaan oleh beberapa peneliti, tetapi peranannya belum banyak diketahui.  Pada penelitian ini dikaji pengaruh penyuntikan hormon estradiol-17β pada perkembangan gonad induk udang putih (Litopenaeus vannamei.  Penelitian ini

  11. Low-frequency, low-magnitude vibrations (LFLM enhances chondrogenic differentiation potential of human adipose derived mesenchymal stromal stem cells (hASCs

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    Krzysztof Marycz

    2016-02-01

    Full Text Available The aim of this study was to evaluate if low-frequency, low-magnitude vibrations (LFLM could enhance chondrogenic differentiation potential of human adipose derived mesenchymal stem cells (hASCs with simultaneous inhibition of their adipogenic properties for biomedical purposes. We developed a prototype device that induces low-magnitude (0.3 g low-frequency vibrations with the following frequencies: 25, 35 and 45 Hz. Afterwards, we used human adipose derived mesenchymal stem cell (hASCS, to investigate their cellular response to the mechanical signals. We have also evaluated hASCs morphological and proliferative activity changes in response to each frequency. Induction of chondrogenesis in hASCs, under the influence of a 35 Hz signal leads to most effective and stable cartilaginous tissue formation through highest secretion of Bone Morphogenetic Protein 2 (BMP-2, and Collagen type II, with low concentration of Collagen type I. These results correlated well with appropriate gene expression level. Simultaneously, we observed significant up-regulation of α3, α4, β1 and β3 integrins in chondroblast progenitor cells treated with 35 Hz vibrations, as well as Sox-9. Interestingly, we noticed that application of 35 Hz frequencies significantly inhibited adipogenesis of hASCs. The obtained results suggest that application of LFLM vibrations together with stem cell therapy might be a promising tool in cartilage regeneration.

  12. The Hypoxia-Mimetic Agent Cobalt Chloride Differently Affects Human Mesenchymal Stem Cells in Their Chondrogenic Potential

    Directory of Open Access Journals (Sweden)

    Gabriella Teti

    2018-01-01

    Full Text Available Adult stem cells are a promising cell source for cartilage regeneration. They resided in a special microenvironment known as the stem-cell niche, characterized by the presence of low oxygen concentration. Cobalt chloride (CoCl2 imitates hypoxia in vitro by stabilizing hypoxia-inducible factor-alpha (HIF-1α, which is the master regulator in the cellular adaptive response to hypoxia. In this study, the influence of CoCl2 on the chondrogenic potential of human MSCs, isolated from dental pulp, umbilical cord, and adipose tissue, was investigated. Cells were treated with concentrations of CoCl2 ranging from 50 to 400 μM. Cell viability, HIF-1α protein synthesis, and the expression of the chondrogenic markers were analyzed. The results showed that the CoCl2 supplementation had no effect on cell viability, while the upregulation of chondrogenic markers such as SOX9, COL2A1, VCAN, and ACAN was dependent on the cellular source. This study shows that hypoxia, induced by CoCl2 treatment, can differently influence the behavior of MSCs, isolated from different sources, in their chondrogenic potential. These findings should be taken into consideration in the treatment of cartilage repair and regeneration based on stem cell therapies.

  13. Mixtures of xenoestrogens disrupt estradiol-induced non-genomic signaling and downstream functions in pituitary cells.

    Science.gov (United States)

    Viñas, René; Watson, Cheryl S

    2013-03-26

    possible apoptotic response. Extrinsic caspase 8 activity was suppressed by estradiol, elevated by bisphenol S, and unaffected by mixtures. Intrinsic caspase 9 activity was inhibited by estradiol, and by xenoestrogen combinations (at 10-14 and 10-8 M). Mixtures of xenoestrogens impeded the estradiol-induced release of prolactin. In mixtures expected to be found in contaminated environments, xenoestrogens can have dramatic disrupting effects on hormonal mechanisms of cell regulation and their downstream functional responses, altering cellular responses to physiologic estrogens.

  14. Enhanced Chondrogenic Differentiation of Human Umbilical Cord Wharton's Jelly Derived Mesenchymal Stem Cells by GSK-3 Inhibitors.

    Directory of Open Access Journals (Sweden)

    Prapot Tanthaisong

    Full Text Available Articular cartilage is an avascular, alymphatic, and aneural system with very low regeneration potential because of its limited capacity for self-repair. Mesenchymal stem cells (MSCs are the preferred choice for cell-based therapies. Glycogen synthase kinase 3 (GSK-3 inhibitors are compounds that can induce the Wnt signaling pathway, which is involved in chondrogenesis and cartilage development. Here, we investigated the influence of lithium chloride (LiCl and SB216763 synergistically with TGF-β3 on chondrogenic differentiation in human mesenchymal stem cells derived from Wharton's jelly tissue (hWJ-MSCs. hWJ-MSCs were cultured and chondrogenic differentiation was induced in monolayer and pellet experiments using chondrogenic medium, chondrogenic medium supplemented with LiCl, or SB216763 for 4 weeks. After in vitro differentiation, cultured cells were examined for the expression of Sox9, ACAN, Col2a1, and β-catenin markers. Glycosaminoglycan (GAG accumulation was also examined by Alcian blue staining. The results indicated that SB216763 was more effective than LiCl as evidenced by a higher up-regulation of the expression of cartilage-specific markers, including Sox9, ACAN, Col2a1 as well as GAG accumulation. Moreover, collagen type II expression was strongly observed in cells cultured in the chondrogenic medium + SB216763 as evidenced by western blot analysis. Both treatments appeared to mediate the Wnt signaling pathway by up-regulating β-catenin gene expression. Further analyses showed that all treatments suppressed the progression of chondrocyte hypertrophy, determined by decreased expression of Col10a1 and Runx2. These results indicate that LiCl and SB216763 are potential candidates for further in vivo therapeutic trials and would be of great importance for cartilage regeneration.

  15. Chondroregulatory action of prolactin on proliferation and differentiation of mouse chondrogenic ATDC5 cells in 3-dimensional micromass cultures

    International Nuclear Information System (INIS)

    Seriwatanachai, Dutmanee; Krishnamra, Nateetip; Charoenphandhu, Narattaphol

    2012-01-01

    Highlights: ► Mouse chondrogenic ATDC5 cells expressed PRL receptor mRNAs and proteins. ► Low PRL concentration (10 ng/mL) increased chondrocyte viability and differentiation. ► Higher PRL concentrations (⩾100 ng/mL) decreased viability and increased apoptosis. -- Abstract: A recent investigation in lactating rats has provided evidence that the lactogenic hormone prolactin (PRL) increases endochondral bone growth and bone elongation, presumably by accelerating apoptosis of hypertrophic chondrocytes in the growth plate and/or subsequent chondrogenic matrix mineralization. Herein, we demonstrated the direct chondroregulatory action of PRL on proliferation, differentiation and apoptosis of chondrocytes in 3-dimensional micromass culture of mouse chondrogenic ATDC5 cell line. The results showed that ATDC5 cells expressed PRL receptor (PRLR) transcripts, and responded typically to PRL by downregulating PRLR expression. Exposure to a low PRL concentration of 10 ng/mL, comparable to the normal levels in male and non-pregnant female rats, increased chondrocyte viability, differentiation, proteoglycan accumulation, and mRNA expression of several chondrogenic differentiation markers, such as Sox9, ALP and Hspg2. In contrast, high PRL concentrations of ⩾100 ng/mL, comparable to the levels in pregnancy or lactation, decreased chondrocyte viability by inducing apoptosis, with no effect on chondrogenic marker expression. It could be concluded that chondrocytes directly but differentially responded to non-pregnant and pregnant/lactating levels of PRL, thus suggesting the stimulatory effect of PRL on chondrogenesis in young growing individuals, and supporting the hypothesis of hypertrophic chondrocyte apoptosis in the growth plate of lactating rats.

  16. Chondroregulatory action of prolactin on proliferation and differentiation of mouse chondrogenic ATDC5 cells in 3-dimensional micromass cultures

    Energy Technology Data Exchange (ETDEWEB)

    Seriwatanachai, Dutmanee [Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Bangkok (Thailand); Krishnamra, Nateetip [Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Bangkok (Thailand); Department of Physiology, Faculty of Science, Mahidol University, Bangkok (Thailand); Charoenphandhu, Narattaphol, E-mail: naratt@narattsys.com [Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Bangkok (Thailand); Department of Physiology, Faculty of Science, Mahidol University, Bangkok (Thailand)

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer Mouse chondrogenic ATDC5 cells expressed PRL receptor mRNAs and proteins. Black-Right-Pointing-Pointer Low PRL concentration (10 ng/mL) increased chondrocyte viability and differentiation. Black-Right-Pointing-Pointer Higher PRL concentrations ( Greater-Than-Or-Slanted-Equal-To 100 ng/mL) decreased viability and increased apoptosis. -- Abstract: A recent investigation in lactating rats has provided evidence that the lactogenic hormone prolactin (PRL) increases endochondral bone growth and bone elongation, presumably by accelerating apoptosis of hypertrophic chondrocytes in the growth plate and/or subsequent chondrogenic matrix mineralization. Herein, we demonstrated the direct chondroregulatory action of PRL on proliferation, differentiation and apoptosis of chondrocytes in 3-dimensional micromass culture of mouse chondrogenic ATDC5 cell line. The results showed that ATDC5 cells expressed PRL receptor (PRLR) transcripts, and responded typically to PRL by downregulating PRLR expression. Exposure to a low PRL concentration of 10 ng/mL, comparable to the normal levels in male and non-pregnant female rats, increased chondrocyte viability, differentiation, proteoglycan accumulation, and mRNA expression of several chondrogenic differentiation markers, such as Sox9, ALP and Hspg2. In contrast, high PRL concentrations of Greater-Than-Or-Slanted-Equal-To 100 ng/mL, comparable to the levels in pregnancy or lactation, decreased chondrocyte viability by inducing apoptosis, with no effect on chondrogenic marker expression. It could be concluded that chondrocytes directly but differentially responded to non-pregnant and pregnant/lactating levels of PRL, thus suggesting the stimulatory effect of PRL on chondrogenesis in young growing individuals, and supporting the hypothesis of hypertrophic chondrocyte apoptosis in the growth plate of lactating rats.

  17. Effects of in vitro low oxygen tension preconditioning of adipose stromal cells on their in vivo chondrogenic potential: application in cartilage tissue repair.

    Directory of Open Access Journals (Sweden)

    Sophie Portron

    Full Text Available PURPOSE: Multipotent stromal cell (MSC-based regenerative strategy has shown promise for the repair of cartilage, an avascular tissue in which cells experience hypoxia. Hypoxia is known to promote the early chondrogenic differentiation of MSC. The aim of our study was therefore to determine whether low oxygen tension could be used to enhance the regenerative potential of MSC for cartilage repair. METHODS: MSC from rabbit or human adipose stromal cells (ASC were preconditioned in vitro in control or chondrogenic (ITS and TGF-β medium and in 21 or 5% O2. Chondrogenic commitment was monitored by measuring COL2A1 and ACAN expression (real-time PCR. Preconditioned rabbit and human ASC were then incorporated into an Si-HPMC hydrogel and injected (i into rabbit articular cartilage defects for 18 weeks or (ii subcutaneously into nude mice for five weeks. The newly formed tissue was qualitatively and quantitatively evaluated by cartilage-specific immunohistological staining and scoring. The phenotype of ASC cultured in a monolayer or within Si-HPMC in control or chondrogenic medium and in 21 or 5% O2 was finally evaluated using real-time PCR. RESULTS/CONCLUSIONS: 5% O2 increased the in vitro expression of chondrogenic markers in ASC cultured in induction medium. Cells implanted within Si-HPMC hydrogel and preconditioned in chondrogenic medium formed a cartilaginous tissue, regardless of the level of oxygen. In addition, the 3D in vitro culture of ASC within Si-HPMC hydrogel was found to reinforce the pro-chondrogenic effects of the induction medium and 5% O2. These data together indicate that although 5% O2 enhances the in vitro chondrogenic differentiation of ASC, it does not enhance their in vivo chondrogenesis. These results also highlight the in vivo chondrogenic potential of ASC and their potential value in cartilage repair.

  18. Investigation of the optimal timing for chondrogenic priming of MSCs to enhance osteogenic differentiation in vitro as a bone tissue engineering strategy.

    Science.gov (United States)

    Freeman, F E; Haugh, M G; McNamara, L M

    2016-04-01

    Recent in vitro tissue engineering approaches have shown that chondrogenic priming of human bone marrow mesenchymal stem cells (MSCs) can have a positive effect on osteogenesis in vivo. However, whether chondrogenic priming is an effective in vitro bone regeneration strategy is not yet known. In particular, the appropriate timing for chondrogenic priming in vitro is unknown albeit that in vivo cartilage formation persists for a specific period before bone formation. The objective of this study is to determine the optimum time for chondrogenic priming of MSCs to enhance osteogenic differentiation by MSCs in vitro. Pellets derived from murine and human MSCs were cultured in six different media groups: two control groups (chondrogenic and osteogenic) and four chondrogenic priming groups (10, 14, 21 and 28 days priming). Biochemical analyses (Hoechst, sulfate glycosaminoglycan (sGAG), Alkaline Phosphate (ALP), calcium), histology (Alcian Blue, Alizarin Red) and immunohistochemistry (collagen types I, II and X) were performed on the samples at specific times. Our results show that after 49 days the highest amount of sGAG production occurred in MSCs chondrogenically primed for 21 days and 28 days. Moreover we found that chondrogenic priming of MSCs in vitro for specific amounts of time (14 days, 21 days) can have optimum influence on their mineralization capacity and can produce a construct that is mineralized throughout the core. Determining the optimum time for chondrogenic priming to enhance osteogenic differentiation in vitro provides information that might lead to a novel regenerative treatment for large bone defects, as well as addressing the major limitation of core degradation and construct failure. Copyright © 2013 John Wiley & Sons, Ltd.

  19. Endometrial safety of ultra-low-dose estradiol vaginal tablets

    DEFF Research Database (Denmark)

    Simon, James; Nachtigall, Lila; Ulrich, Lian G

    2010-01-01

    To evaluate the endometrial hyperplasia and carcinoma rate after 52-week treatment with ultra-low-dose 10-microgram 17ß-estradiol vaginal tablets in postmenopausal women with vaginal atrophy.......To evaluate the endometrial hyperplasia and carcinoma rate after 52-week treatment with ultra-low-dose 10-microgram 17ß-estradiol vaginal tablets in postmenopausal women with vaginal atrophy....

  20. Endometrial safety of ultra-low-dose estradiol vaginal tablets

    DEFF Research Database (Denmark)

    Simon, James; Nachtigall, Lila; Ulrich, Lian G

    2010-01-01

    To evaluate the endometrial hyperplasia and carcinoma rate after 52-week treatment with ultra-low-dose 10-microgram 17β-estradiol vaginal tablets in postmenopausal women with vaginal atrophy.......To evaluate the endometrial hyperplasia and carcinoma rate after 52-week treatment with ultra-low-dose 10-microgram 17β-estradiol vaginal tablets in postmenopausal women with vaginal atrophy....

  1. Hypoxic chondrogenic differentiation of human cord blood stem cells in structurally-graded polycaprolactone scaffolds

    DEFF Research Database (Denmark)

    Munir, Samir; Søballe, Kjeld; Ulrich-Vinther, Michael

    culturing resulted in a multicellular layer tissue with formation of more cartilaginous tissue compared to micromass or CPP culture. In the membrane system MLPCs produced pellucid discs, 12 mm in diameter by 1 mm in thickness from 2x10^6 cells. The discs had hyaline-like cartilage extracellular matrix......Background: Articular chondrocytes and bone marrow-derived multipotent mesenchymal stromal cells (MSCs) are the favoured cells for cartilage tissue engineering. Umbilical cord blood has proven an alternative source of MSCs and moreover they may be more potent chondroprogenitor cells than bonemarrow...... with micromass or CPP cultures. Conclusions: In conclusion, we demonstrate that MLPCs possess’ chondrogenic potency, which increased when cultured scaffold-free on membrane inserts resulting in multicellular-layered hyaline-like cartilage tissue. Evaluating the effect of culturing pre-differentiated MLPCs on CPP...

  2. Interaction of estradiol and high density lipoproteins on proliferation of the human breast cancer cell line MCF-7 adapted to grow in serum free conditions

    International Nuclear Information System (INIS)

    Jozan, S.; Faye, J.C.; Tournier, J.F.; Tauber, J.P.; David, J.F.; Bayard, F.

    1985-01-01

    The responsiveness of the human mammary carcinoma cell line MCF-7 to estradiol and tamoxifen treatment has been studied in different culture conditions. Cells from exponentially growing cultures were compared with cells in their initial cycles after replating from confluent cultures (''confluent-log'' cells). It has been observed that estradiol stimulation of tritiated thymidine incorporation decreases with cell density and that ''confluent-log'' cells are estrogen unresponsive for a period of four cell cycles in serum-free medium conditions. On the other hand, growth of cells replated from exponentially growing, as well as from confluent cultures, can be inhibited by tamoxifen or a combined treatment with tamoxifen and the progestin levonorgestrel. This growth inhibitory effect can be rescued by estradiol when cells are replated from exponentially growing cultures. The growth inhibitory effect cannot be rescued by estradiol alone (10(-10) to 10(-8) M) when cells are replated from confluent cultures. In this condition, the addition of steroid depleted serum is necessary to reverse the state of estradiol unresponsiveness. Serum can be replaced by high density lipoproteins but not by low density lipoproteins or lipoprotein deficient serum. The present data show that estradiol and HDL interact in the control of MCF-7 cell proliferation

  3. Indian hedgehog gene transfer is a chondrogenic inducer of human mesenchymal stem cells

    Science.gov (United States)

    2012-01-01

    Introduction To date, no single most-appropriate factor or delivery method has been identified for the purpose of mesenchymal stem cell (MSC)-based treatment of cartilage injury. Therefore, in this study we tested whether gene delivery of the growth factor Indian hedgehog (IHH) was able to induce chondrogenesis in human primary MSCs, and whether it was possible by such an approach to modulate the appearance of chondrogenic hypertrophy in pellet cultures in vitro. Methods First-generation adenoviral vectors encoding the cDNA of the human IHH gene were created by cre-lox recombination and used alone or in combination with adenoviral vectors, bone morphogenetic protein-2 (Ad.BMP-2), or transforming growth factor beta-1 (Ad.TGF-β1) to transduce human bone-marrow derived MSCs at 5 × 102 infectious particles/cell. Thereafter, 3 × 105 cells were seeded into aggregates and cultured for 3 weeks in serum-free medium, with untransduced or marker gene transduced cultures as controls. Transgene expressions were determined by ELISA, and aggregates were analysed histologically, immunohistochemically, biochemically and by RT-PCR for chondrogenesis and hypertrophy. Results IHH, TGF-β1 and BMP-2 genes were equipotent inducers of chondrogenesis in primary MSCs, as evidenced by strong staining for proteoglycans, collagen type II, increased levels of glycosaminoglycan synthesis, and expression of mRNAs associated with chondrogenesis. IHH-modified aggregates, alone or in combination, also showed a tendency to progress towards hypertrophy, as judged by the expression of alkaline phosphatase and stainings for collagen type X and Annexin 5. Conclusion As this study provides evidence for chondrogenic induction of MSC aggregates in vitro via IHH gene delivery, this technology may be efficiently employed for generating cartilaginous repair tissues in vivo. PMID:22817660

  4. Chondrogenic differentiation of ATDC5-cells under the influence of Mg and Mg alloy degradation.

    Science.gov (United States)

    Martinez Sanchez, Adela H; Feyerabend, Frank; Laipple, Daniel; Willumeit-Römer, Regine; Weinberg, Annelie; Luthringer, Bérengère J C

    2017-03-01

    Biodegradable magnesium (Mg)-based materials are a potential alternative to permanent implants for application in children. Nevertheless effects of those materials on growth plate cartilage and chondrogenesis have not been previously evaluated. In vitro differentiation of ATDC5 cells was evaluated under the influence of pure Mg (PMg), Mg with 10wt% of gadolinium (Mg-10Gd) and Mg with 2wt% of silver (Mg-2Ag) degradation products (extracts) and direct cell culture on the materials. Gene expression showed an inhibitory effect on ATDC5 mineralization with the three extracts and a chondrogenic potential of Mg-10Gd. Cells cultured in Mg-10Gd and Mg-2Ag extracts showed the same proliferation and morphology than cells cultured in growth conditions. Mg-10Gd induced an increase in production of ECM and a bigger cell size, similar to the effects found with differentiation conditions. An increased metabolic activity was observed in cells cultured under the influence of Mg-10Gd extracts, indicated by an acidic pH during most of the culture period. After 7days of culture on the materials, ATDC5 growth, distribution and ECM synthesis were higher on Mg-10Gd samples, followed by Mg-2Ag and PMg, which was influenced by the homogeneity and composition of the degradation layer. This study confirmed the tolerance of ATDC5 cells to Mg-based materials and a chondrogenic effect of Mg-10Gd. Further studies in vitro and in vivo are necessary to evaluate cell reactions to those materials, as well as the effects on bone growth and the biocompatibility of the alloying system in the body. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Relationship between Estradiol and Antioxidant Enzymes Activity of Ischemic Stroke

    Directory of Open Access Journals (Sweden)

    Nasrin Sheikh

    2009-01-01

    Full Text Available Some evidence suggests the neuroprotection of estrogen provided by the antioxidant activity of this compound. The main objective of this study was to determine the level of estradiol and its correlation with the activity of antioxidant enzymes, total antioxidant status and ferritin from ischemic stroke subjects. The study population consisted of 30 patients with acute ischemic stroke and 30 controls. There was no significant difference between estradiol in stroke and control group. The activity of superoxide dismutase and level of ferritin was higher in stroke compared with control group (<.05, <.001, resp.. There was no significant correlation between estradiol and glutathione peroxidase, glutathione reductase, catalase, total antioxidant status, and ferritin in stroke and control groups. We observed inverse correlation between estradiol with superoxide dismutase in males of stroke patients (=−0.54, =.029. Our results supported that endogenous estradiol of elderly men and women of stroke or control group has no antioxidant activity.

  6. Why use of dienogest for the first contraceptive pill with estradiol?

    Science.gov (United States)

    Mueck, Alfred O; Seeger, Harald; Bühling, Kai J

    2010-02-01

    Dienogest (DNG) has the essential properties of an effective progestogen for use in a new contraceptive pill using estradiol valerate as estrogenic component -- it inhibits ovulation and protects against endometrial proliferation. DNG is a derivative of norethisterone (NET), but has a cyanomethyl- instead of an ethinyl-group in C17 position which may offer a variety of benefits regarding hepatic effects. The similarity to NET is reflected in the high endometriotropy and in similar pharmacokinetics like short plasma half-live and high bioavailability. However, DNG also elicits properties of progesterone derivatives like neutrality in metabolic and cardiovascular system and considerable antiandrogenic activity, the latter increased by lack of binding to SHBG as specific property of DNG. It has no glucocorticoid and antimineralocorticoid activity and has no antiestrogenic activity with the consequence that possible beneficial estradiol effects should not be antagonized. This may be of special importance for the tolerability and safety of the first pill with estradiol valerate instead of ethinylestradiol, although well-designed postmarketing studies are still ongoing to demonstrate what can be expected on the basis of pharmacology.

  7. Estradiol-induced estrogen receptor-alpha trafficking.

    Science.gov (United States)

    Bondar, Galyna; Kuo, John; Hamid, Naheed; Micevych, Paul

    2009-12-02

    Estradiol has rapid actions in the CNS that are mediated by membrane estrogen receptors (ERs) and activate cell signaling pathways through interaction with metabotropic glutamate receptors (mGluRs). Membrane-initiated estradiol signaling increases the free cytoplasmic calcium concentration ([Ca(2+)](i)) that stimulates the synthesis of neuroprogesterone in astrocytes. We used surface biotinylation to demonstrate that ERalpha has an extracellular portion. In addition to the full-length ERalpha [apparent molecular weight (MW), 66 kDa], surface biotinylation labeled an ERalpha-immunoreactive protein (MW, approximately 52 kDa) identified by both COOH- and NH(2)-directed antibodies. Estradiol treatment regulated membrane levels of both proteins in parallel: within 5 min, estradiol significantly increased membrane levels of the 66 and 52 kDa ERalpha. Internalization, a measure of membrane receptor activation, was also increased by estradiol with a similar time course. Continuous treatment with estradiol for 24-48 h reduced ERalpha levels, suggesting receptor downregulation. Estradiol also increased mGluR1a trafficking and internalization, consistent with the proposed ERalpha-mGluR1a interaction. Blocking ER with ICI 182,780 or mGluR1a with LY 367385 prevented ERalpha trafficking to and from the membrane. Estradiol-induced [Ca(2+)](i) flux was also significantly increased at the time of peak ERalpha activation/internalization. These results demonstrate that ERalpha is present in the membrane and has an extracellular portion. Furthermore, membrane levels and internalization of ERalpha are regulated by estradiol and mGluR1a ligands. The pattern of trafficking into and out of the membrane suggests that the changing concentration of estradiol during the estrous cycle regulates ERalpha to augment and then terminate membrane-initiated signaling.

  8. Estradiol-induced estrogen receptor-α trafficking

    Science.gov (United States)

    Bondar, Galyna; Kuo, John; Hamid, Naheed; Micevych, Paul

    2010-01-01

    Estradiol has rapid actions in the central nervous system, which are mediated by membrane estrogen receptors (ERs) and activate cell signaling pathways through interaction with metabotropic glutamate receptors (mGluRs). Membrane-initiated estradiol signaling increases the free cytoplasmic calcium concentration ([Ca2+]i) that stimulates the synthesis of neuroprogesterone in astrocytes. We used surface biotinylation to demonstrate that ERα has an extracellular portion. In addition to the full length ERα (apparent M.W. 66 kDa), surface biotinylation labeled an ERα-immunoreactive protein (M.W. ~ 52 kDa) identified by both COOH- and NH2-directed antibodies. Estradiol treatment regulated membrane levels of both proteins in parallel: within 5 min, estradiol significantly increased membrane levels of the 66 kDa and 52 kDa ERα. Internalization, a measure of membrane receptor activation, was also increased by estradiol with a similar time course. Continuous treatment with estradiol for 24–48 hr reduced ERα levels, suggesting receptor down-regulation. Estradiol also increased mGluR1a trafficking and internalization, consistent with the proposed ERα-mGluR1a interaction. Blocking ER with ICI 182,780 or mGluR1a with LY 367385 prevented ERα trafficking to and from the membrane. Estradiol-induced [Ca2+]i flux was also significantly increased at the time of peak ERα activation/internalization. These results demonstrate that ERα is present in the membrane and has an extracellular portion. Furthermore, membrane levels and internalization of ERα are regulated by estradiol and mGluR1a ligands. The pattern of trafficking into and out of the membrane suggests that the changing concentration of estradiol during the estrous cycle regulates ERα to augment and then terminate membrane-initiated signaling. PMID:19955385

  9. Estradiol and luteinizing hormone regulate recognition memory following subchronic phencyclidine: Evidence for hippocampal GABA action.

    Science.gov (United States)

    Riordan, Alexander J; Schaler, Ari W; Fried, Jenny; Paine, Tracie A; Thornton, Janice E

    2018-05-01

    The cognitive symptoms of schizophrenia are poorly understood and difficult to treat. Estrogens may mitigate these symptoms via unknown mechanisms. To examine these mechanisms, we tested whether increasing estradiol (E) or decreasing luteinizing hormone (LH) could mitigate short-term episodic memory loss in a phencyclidine (PCP) model of schizophrenia. We then assessed whether changes in cortical or hippocampal GABA may underlie these effects. Female rats were ovariectomized and injected subchronically with PCP. To modulate E and LH, animals received estradiol capsules or Antide injections. Short-term episodic memory was assessed using the novel object recognition task (NORT). Brain expression of GAD67 was analyzed via western blot, and parvalbumin-containing cells were counted using immunohistochemistry. Some rats received hippocampal infusions of a GABA A agonist, GABA A antagonist, or GAD inhibitor before behavioral testing. We found that PCP reduced hippocampal GAD67 and abolished recognition memory. Antide restored hippocampal GAD67 and rescued recognition memory in PCP-treated animals. Estradiol prevented PCP's amnesic effect in NORT but failed to restore hippocampal GAD67. PCP did not cause significant differences in number of parvalbumin-expressing cells or cortical expression of GAD67. Hippocampal infusions of a GABA A agonist restored recognition memory in PCP-treated rats. Blocking hippocampal GAD or GABA A receptors in ovx animals reproduced recognition memory loss similar to PCP and inhibited estradiol's protection of recognition memory in PCP-treated animals. In summary, decreasing LH or increasing E can lessen short-term episodic memory loss, as measured by novel object recognition, in a PCP model of schizophrenia. Alterations in hippocampal GABA may contribute to both PCP's effects on recognition memory and the hormones' ability to prevent or reverse them. Copyright © 2018 Elsevier Ltd. All rights reserved.

  10. Estrogen inhibits chloride secretion caused by cholera and Escherichia coli enterotoxins in female rat distal colon.

    LENUS (Irish Health Repository)

    Alzamora, Rodrigo

    2011-05-08

    Excessive Cl(-) secretion is the driving force for secretory diarrhea. 17β-Estradiol has been shown to inhibit Cl(-) secretion in rat distal colon through a nongenomic pathway. We examined whether 17β-estradiol inhibits Cl(-) secretion in an animal model of secretory diarrhea and the downstream effectors involved. The effect of 17β-estradiol on cholera toxin and heat-stable enterotoxin induced Cl(-) secretion in rat colonic mucosal sheets was studied by current-voltage clamping. Selective permeabilization of apical or basolateral membranes with amphotericin B or nystatin was used to isolate basolateral K(+) channel and apical Cl(-) channel activity, respectively. 17β-Estradiol dose-dependently inhibited secretory responses to both toxins with IC(50) values of approximately 1nM. This effect was female-gender specific, with no inhibition observed in male tissues. 17β-Estradiol responses were insensitive to the pure anti-estrogen ICI 182,720. 17β-Estradiol exerted its effects downstream of enterotoxin-induced production of second messengers (cAMP and cGMP) but was dependent on PKCδ activation. In nystatin-permeabilized tissues, apical Cl(-) currents were unaffected by 17β-estradiol treatment while basolateral K(+) current was profoundly inhibited by the hormone. This current was sensitive to the specific KCNQ1 channel inhibitors chromanol 293B and HMR-1556. In conclusion, 17β-estradiol inhibits enterotoxin-induced Cl(-) secretion via a PKCδ-dependent mechanism involving inhibition of basolateral KCNQ1 channels. These data elucidate mechanisms of 17β-estradiol inhibition of Cl(-) secretion induced by enterotoxins in intestinal epithelia, which may be relevant for the treatment of diarrheal diseases.

  11. Estrogen inhibits chloride secretion caused by cholera and Escherichia coli enterotoxins in female rat distal colon.

    LENUS (Irish Health Repository)

    Alzamora, Rodrigo

    2012-02-01

    Excessive Cl(-) secretion is the driving force for secretory diarrhea. 17beta-Estradiol has been shown to inhibit Cl(-) secretion in rat distal colon through a nongenomic pathway. We examined whether 17beta-estradiol inhibits Cl(-) secretion in an animal model of secretory diarrhea and the downstream effectors involved. The effect of 17beta-estradiol on cholera toxin and heat-stable enterotoxin induced Cl(-) secretion in rat colonic mucosal sheets was studied by current-voltage clamping. Selective permeabilization of apical or basolateral membranes with amphotericin B or nystatin was used to isolate basolateral K(+) channel and apical Cl(-) channel activity, respectively. 17beta-Estradiol dose-dependently inhibited secretory responses to both toxins with IC(50) values of approximately 1nM. This effect was female-gender specific, with no inhibition observed in male tissues. 17beta-Estradiol responses were insensitive to the pure anti-estrogen ICI 182,720. 17beta-Estradiol exerted its effects downstream of enterotoxin-induced production of second messengers (cAMP and cGMP) but was dependent on PKCdelta activation. In nystatin-permeabilized tissues, apical Cl(-) currents were unaffected by 17beta-estradiol treatment while basolateral K(+) current was profoundly inhibited by the hormone. This current was sensitive to the specific KCNQ1 channel inhibitors chromanol 293B and HMR-1556. In conclusion, 17beta-estradiol inhibits enterotoxin-induced Cl(-) secretion via a PKCdelta-dependent mechanism involving inhibition of basolateral KCNQ1 channels. These data elucidate mechanisms of 17beta-estradiol inhibition of Cl(-) secretion induced by enterotoxins in intestinal epithelia, which may be relevant for the treatment of diarrheal diseases.

  12. MiR-193b regulates early chondrogenesis by inhibiting the TGF-beta2 signaling pathway.

    Science.gov (United States)

    Hou, Changhe; Yang, Zibo; Kang, Yan; Zhang, Ziji; Fu, Ming; He, Aishan; Zhang, Zhiqi; Liao, Weiming

    2015-04-13

    Cartilage generation and degradation are regulated by miRNAs. Our previous study has shown altered expression of miR-193b in chondrogenic human adipose-derived mesenchymal stem cells (hADSCs). In the current study, we investigated the role of miR-193b in chondrogenesis and cartilage degradation. Luciferase reporter assays showed that miR-193b targeted seed sequences of the TGFB2 and TGFBR3 3'-UTRs. MiR-193b suppressed the expression of early chondrogenic markers in chondrogenic ATDC5 cells, and TNF-alpha expression in IL-1b-induced PMCs. In conclusion, MiR-193b may inhibit early chondrogenesis by targeting TGFB2 and TGFBR3, and may regulate inflammation by repressing TNF-alpha expression in inflamed chondrocytes. Copyright © 2015. Published by Elsevier B.V.

  13. Mechanisms of estradiol-induced insulin secretion by the G protein-coupled estrogen receptor GPR30/GPER in pancreatic beta-cells.

    Science.gov (United States)

    Sharma, Geetanjali; Prossnitz, Eric R

    2011-08-01

    Sexual dimorphism and supplementation studies suggest an important role for estrogens in the amelioration of glucose intolerance and diabetes. Because little is known regarding the signaling mechanisms involved in estradiol-mediated insulin secretion, we investigated the role of the G protein-coupled receptor 30, now designated G protein-coupled estrogen receptor (GPER), in activating signal transduction cascades in β-cells, leading to secretion of insulin. GPER function in estradiol-induced signaling in the pancreatic β-cell line MIN6 was assessed using small interfering RNA and GPER-selective ligands (G-1 and G15) and in islets isolated from wild-type and GPER knockout mice. GPER is expressed in MIN6 cells, where estradiol and the GPER-selective agonist G-1 mediate calcium mobilization and activation of ERK and phosphatidylinositol 3-kinase. Both estradiol and G-1 induced insulin secretion under low- and high-glucose conditions, which was inhibited by pretreatment with GPER antagonist G15 as well as depletion of GPER by small interfering RNA. Insulin secretion in response to estradiol and G-1 was dependent on epidermal growth factor receptor and ERK activation and further modulated by phosphatidylinositol 3-kinase activity. In islets isolated from wild-type mice, the GPER antagonist G15 inhibited insulin secretion induced by estradiol and G-1, both of which failed to induce insulin secretion in islets obtained from GPER knockout mice. Our results indicate that GPER activation of the epidermal growth factor receptor and ERK in response to estradiol treatment plays a critical role in the secretion of insulin from β-cells. The results of this study suggest that the activation of downstream signaling pathways by the GPER-selective ligand G-1 could represent a novel therapeutic strategy in the treatment of diabetes.

  14. Mechanisms of Estradiol-Induced Insulin Secretion by the G Protein-Coupled Estrogen Receptor GPR30/GPER in Pancreatic β-Cells

    Science.gov (United States)

    Sharma, Geetanjali

    2011-01-01

    Sexual dimorphism and supplementation studies suggest an important role for estrogens in the amelioration of glucose intolerance and diabetes. Because little is known regarding the signaling mechanisms involved in estradiol-mediated insulin secretion, we investigated the role of the G protein-coupled receptor 30, now designated G protein-coupled estrogen receptor (GPER), in activating signal transduction cascades in β-cells, leading to secretion of insulin. GPER function in estradiol-induced signaling in the pancreatic β-cell line MIN6 was assessed using small interfering RNA and GPER-selective ligands (G-1 and G15) and in islets isolated from wild-type and GPER knockout mice. GPER is expressed in MIN6 cells, where estradiol and the GPER-selective agonist G-1 mediate calcium mobilization and activation of ERK and phosphatidylinositol 3-kinase. Both estradiol and G-1 induced insulin secretion under low- and high-glucose conditions, which was inhibited by pretreatment with GPER antagonist G15 as well as depletion of GPER by small interfering RNA. Insulin secretion in response to estradiol and G-1 was dependent on epidermal growth factor receptor and ERK activation and further modulated by phosphatidylinositol 3-kinase activity. In islets isolated from wild-type mice, the GPER antagonist G15 inhibited insulin secretion induced by estradiol and G-1, both of which failed to induce insulin secretion in islets obtained from GPER knockout mice. Our results indicate that GPER activation of the epidermal growth factor receptor and ERK in response to estradiol treatment plays a critical role in the secretion of insulin from β-cells. The results of this study suggest that the activation of downstream signaling pathways by the GPER-selective ligand G-1 could represent a novel therapeutic strategy in the treatment of diabetes. PMID:21673097

  15. The trans-well coculture of human synovial mesenchymal stem cells with chondrocytes leads to self-organization, chondrogenic differentiation, and secretion of TGFβ

    DEFF Research Database (Denmark)

    Kubosch, Eva Johanna; Heidt, Emanuel; Bernstein, Anke

    2016-01-01

    BACKGROUND: Synovial mesenchymal stem cells (SMSC) possess a high chondrogenic differentiation potential, which possibly supports natural and surgically induced healing of cartilage lesions. We hypothesized enhanced chondrogenesis of SMSC caused by the vicinity of chondrocytes (CHDR). METHODS...

  16. Preparation of 16β-Estradiol Derivative Libraries as Bisubstrate Inhibitors of 17β-Hydroxysteroid Dehydrogenase Type 1 Using the Multidetachable Sulfamate Linker

    Directory of Open Access Journals (Sweden)

    Donald Poirier

    2010-03-01

    Full Text Available Combinatorial chemistry is a powerful tool used to rapidly generate a large number of potentially biologically active compounds. In our goal to develop bisubstrate inhibitors of 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1 that interact with both the substrate (estrone or estradiol and the cofactor (NAD(PH binding sites, we used parallel solid-phase synthesis to prepare three libraries of 16β-estradiol derivatives with two or three levels of molecular diversity. From estrone, we first synthesized a sulfamate precursor that we loaded on trityl chloride resin using the efficient multidetachable sulfamate linker strategy recently developed in our laboratory. We then introduced molecular diversity [one or two amino acid(s followed by a carboxylic acid] on steroid nucleus by Fmoc peptide chemistry. Finally, after a nucleophilic cleavage, libraries of 30, 63 and 25 estradiol derivatives were provided. A library of 30 sulfamoylated estradiol derivatives was also generated by acidic cleavage and its members were screened for inhibition of steroid sulfatase. Biological evaluation on homogenated HEK-293 cells overexpressing 17β-HSD1 of the estradiol derivatives carrying different oligoamide-type chains at C-16 first revealed that three levels of molecular diversity (a spacer of two amino acids were necessary to interact with the adenosine part of the cofactor binding site. Second, the best inhibition was obtained when hydrophobic residues (phenylalanine were used as building blocks.

  17. CONCENTRATION OF ESTRADIOL IN DOGS (BITCHES IN SPRINGTIME

    Directory of Open Access Journals (Sweden)

    Edina Hajdarević

    2013-09-01

    Full Text Available Measuring of estradiol level in the peripheral blood in dog is important for the precise estrus detection. In proestrus, estradiol dominates. In estrus, however, estradiol progressively decreases while progesterone and LH increase, the latter shortly and abruptly. The research of Feldman and Nelson (7 indicates that the beginning of the sexual cycle of the female dog is the result of complex interaction of the environment, general health condition, condition of the ovaries, condition of the uterus, animal age, and some unidentified factors. Estradiol level in the peripheral circulation is starting to rise before the beginning of the proestrus, and during the proestrus the female dog is under the influence of estradiol (4. Our research included 30 female dogs on the territory of Tuzla Municipality, in the springtime. The female dogs were divided in three groups according to the breeding and living conditions: group A (female dogs living in the house environment; group B (female dogs living in the shelter; group C (female stray dogs. For the researched groups, estradiol level varied between 6,265 pg\\ml and 69,734 pg\\ml over the springtime. Of importance is the results can be applied in the evaluation of estrus in the female dogs, and when considering factors crucial for their sustainable reproduction potential.Key words: dogs, estradiol, spring

  18. Involvement of CART in estradiol-induced anorexia.

    Science.gov (United States)

    Dandekar, Manoj P; Nakhate, Kartik T; Kokare, Dadasaheb M; Subhedar, Nishikant K

    2012-01-18

    Since estradiol exercises inhibitory effect on food intake, we wanted to find out if this influence of estradiol is mediated by cocaine- and amphetamine-regulated transcript peptide (CART), a well established anorectic agent in the brain. Ovariectomized (OVX) rats, replaced with estradiol to produce estrous-phase like conditions, showed a significant decrease in food intake as compared with that in OVX controls. Intracerebroventricular (icv) administration of CART (0.5-1 μg/rat) to OVX rats, resulted in a dose-dependent reduction in the food intake. The lower dose (0.25 μg) had no effect, and was considered subeffective. In estradiol replaced OVX rats, CART at subeffective dose, further reduced food intake. However, CART failed to reduce food intake in estradiol replaced OVX rats pretreated with anti-estrogenic agent tamoxifen (3 mg/kg, subcutaneous). Administration of CART antibody (1:500 dilution/rat, i.c.v.) significantly attenuated estradiol-induced anorexia in the OVX rats. While estradiol replacement significantly increased CART-immunoreactivity in the cells/fibers of paraventricular nucleus (PVN) of OVX rats, fibers in the anteroventral periventricular nucleus (AVPV), and cells/fibers in the arcuate nucleus (ARC) showed considerable reduction. These changes were attenuated following concurrent injection of tamoxifen to the estradiol replaced OVX rats. However, CART-immunoreactive cells/fibers in the periventricular area did not respond to any of the treatments. We suggest that estradiol treatment might influence the hypothalamic CART system in a site specific manner. While increased CART activity in the PVN might produce anorexia, reduction of CART in ARC and AVPV might represent a compensatory homeostatic response. Copyright © 2011 Elsevier Inc. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    2014-01-01

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Yong Il; Ryu, Jae-Sung; Yeo, Jee Eun; Choi, Yun Jin; Kim, Yong Sang [Center for Stem Cell and Arthritis Research, Department of Orthopedic Surgery, Yonsei Sarang Hospital, Seoul (Korea, Republic of); Ko, Kinarm [Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University, Seoul 143-701 (Korea, Republic of); Koh, Yong-Gon, E-mail: yonseranglab@daum.net [Center for Stem Cell and Arthritis Research, Department of Orthopedic Surgery, Yonsei Sarang Hospital, Seoul (Korea, Republic of)

    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.

  1. Chondrogenic Differentiation of Defined Equine Mesenchymal Stem Cells Derived from Umbilical Cord Blood for Use in Cartilage Repair Therapy

    Directory of Open Access Journals (Sweden)

    Mélanie Desancé

    2018-02-01

    Full Text Available Cartilage engineering is a new strategy for the treatment of cartilage damage due to osteoarthritis or trauma in humans. Racehorses are exposed to the same type of cartilage damage and the anatomical, cellular, and biochemical properties of their cartilage are comparable to those of human cartilage, making the horse an excellent model for the development of cartilage engineering. Human mesenchymal stem cells (MSCs differentiated into chondrocytes with chondrogenic factors in a biomaterial appears to be a promising therapeutic approach for direct implantation and cartilage repair. Here, we characterized equine umbilical cord blood-derived MSCs (eUCB-MSCs and evaluated their potential for chondrocyte differentiation for use in cartilage repair therapy. Our results show that isolated eUCB-MSCs had high proliferative capacity and differentiated easily into osteoblasts and chondrocytes, but not into adipocytes. A three-dimensional (3D culture approach with the chondrogenic factors BMP-2 and TGF-β1 potentiated chondrogenic differentiation with a significant increase in cartilage-specific markers at the mRNA level (Col2a1, Acan, Snorc and the protein level (type II and IIB collagen without an increase in hypertrophic chondrocyte markers (Col10a1 and Mmp13 in normoxia and in hypoxia. However, these chondrogenic factors caused an increase in type I collagen, which can be reduced using small interfering RNA targeting Col1a2. This study provides robust data on MSCs characterization and demonstrates that eUCB-MSCs have a great potential for cartilage tissue engineering.

  2. Estradiol and endocrine disrupting compounds adversely affect development of sea urchin embryos at environmentally relevant concentrations

    International Nuclear Information System (INIS)

    Roepke, Troy A.; Snyder, Mark J.; Cherr, Gary N.

    2005-01-01

    Environmental endocrine disrupting compounds (EDCs) are a wide variety of chemicals that typically exert effects, either directly or indirectly, through receptor-mediated processes, thus mimicking endogenous hormones and/or inhibiting normal hormone activities and metabolism. Little is known about the effects of EDCs on echinoderm physiology, reproduction and development. We exposed developing sea urchin embryos (Strongylocentrotus purpuratus and Lytechinus anamesus) to two known EDCs (4-octylphenol (OCT), bisphenol A (BisA)) and to natural and synthetic reproductive hormones (17β-estradiol (E 2 ), estrone (E 1 ), estriol (E 3 ), progesterone (P 4 ) and 17α-ethynylestradiol (EE 2 )). In addition, we studied two non-estrogenic EDCs, tributyltin (TBT) and o,p-DDD. Successful development to the pluteus larval stage (96 h post-fertilization) was used to define EDC concentration-response relationships. The order of compound potency based on EC 50 values for a reduction in normal development was as follows: TBT L.anamesus > OCT > TBT S. p urpuratus >> E 2 > EE 2 > DDD >> BisA > P 4 > E 1 >> E 3 . The effect of TBT was pronounced even at concentrations substantially lower than those commonly reported in heavily contaminated areas, but the response was significantly different in the two model species. Sea urchin embryos were generally more sensitive to estrogenic EDCs and TBT than most other invertebrate larvae. Stage-specific exposure experiments were conducted to determine the most sensitive developmental periods using blastula, gastrula and post-gastrula (pluteus) stages. The stage most sensitive to E 2 , OCT and TBT was the blastula stage with less overall sensitivity in the gastrula stage, regardless of concentration. Selective estrogen receptor modulators (SERMs) were added to the experiments individually and in combination with estrogenic EDCs to interfere with potential receptor-mediated actions. Tamoxifen, a partial ER agonist, alone inhibited development at

  3. The changing integrin expression and a role for integrin β8 in the chondrogenic differentiation of mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Vanessa L S LaPointe

    Full Text Available Many cartilage tissue engineering approaches aim to differentiate human mesenchymal stem cells (hMSCs into chondrocytes and develop cartilage in vitro by targeting cell-matrix interactions. We sought to better inform the design of cartilage tissue engineering scaffolds by understanding how integrin expression changes during chondrogenic differentiation. In three models of in vitro chondrogenesis, we studied the temporal change of cartilage phenotype markers and integrin subunits during the differentiation of hMSCs. We found that transcript expression of most subunits was conserved across the chondrogenesis models, but was significantly affected by the time-course of differentiation. In particular, ITGB8 was up-regulated and its importance in chondrogenesis was further established by a knockdown of integrin β8, which resulted in a non-hyaline cartilage phenotype, with no COL2A1 expression detected. In conclusion, we performed a systematic study of the temporal changes of integrin expression during chondrogenic differentiation in multiple chondrogenesis models, and revealed a role for integrin β8 in chondrogenesis. This work enhances our understanding of the changing adhesion requirements of hMSCs during chondrogenic differentiation and underlines the importance of integrins in establishing a cartilage phenotype.

  4. The Changing Integrin Expression and a Role for Integrin β8 in the Chondrogenic Differentiation of Mesenchymal Stem Cells

    Science.gov (United States)

    LaPointe, Vanessa L. S.; Verpoorte, Amanda; Stevens, Molly M.

    2013-01-01

    Many cartilage tissue engineering approaches aim to differentiate human mesenchymal stem cells (hMSCs) into chondrocytes and develop cartilage in vitro by targeting cell-matrix interactions. We sought to better inform the design of cartilage tissue engineering scaffolds by understanding how integrin expression changes during chondrogenic differentiation. In three models of in vitro chondrogenesis, we studied the temporal change of cartilage phenotype markers and integrin subunits during the differentiation of hMSCs. We found that transcript expression of most subunits was conserved across the chondrogenesis models, but was significantly affected by the time-course of differentiation. In particular, ITGB8 was up-regulated and its importance in chondrogenesis was further established by a knockdown of integrin β8, which resulted in a non-hyaline cartilage phenotype, with no COL2A1 expression detected. In conclusion, we performed a systematic study of the temporal changes of integrin expression during chondrogenic differentiation in multiple chondrogenesis models, and revealed a role for integrin β8 in chondrogenesis. This work enhances our understanding of the changing adhesion requirements of hMSCs during chondrogenic differentiation and underlines the importance of integrins in establishing a cartilage phenotype. PMID:24312400

  5. Monomeric, porous type II collagen scaffolds promote chondrogenic differentiation of human bone marrow mesenchymal stem cells in vitro

    Science.gov (United States)

    Tamaddon, M.; Burrows, M.; Ferreira, S. A.; Dazzi, F.; Apperley, J. F.; Bradshaw, A.; Brand, D. D.; Czernuszka, J.; Gentleman, E.

    2017-03-01

    Osteoarthritis (OA) is a common cause of pain and disability and is often associated with the degeneration of articular cartilage. Lesions to the articular surface, which are thought to progress to OA, have the potential to be repaired using tissue engineering strategies; however, it remains challenging to instruct cell differentiation within a scaffold to produce tissue with appropriate structural, chemical and mechanical properties. We aimed to address this by driving progenitor cells to adopt a chondrogenic phenotype through the tailoring of scaffold composition and physical properties. Monomeric type-I and type-II collagen scaffolds, which avoid potential immunogenicity associated with fibrillar collagens, were fabricated with and without chondroitin sulfate (CS) and their ability to stimulate the chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells was assessed. Immunohistochemical analyses showed that cells produced abundant collagen type-II on type-II scaffolds and collagen type-I on type-I scaffolds. Gene expression analyses indicated that the addition of CS - which was released from scaffolds quickly - significantly upregulated expression of type II collagen, compared to type-I and pure type-II scaffolds. We conclude that collagen type-II and CS can be used to promote a more chondrogenic phenotype in the absence of growth factors, potentially providing an eventual therapy to prevent OA.

  6. Enhancement of chondrogenic differentiation of rabbit mesenchymal stem cells by oriented nanofiber yarn-collagen type I/hyaluronate hybrid

    International Nuclear Information System (INIS)

    Zheng, Xianyou; Wang, Wei; Liu, Shen; Wu, Jinglei; Li, Fengfeng; Cao, Lei; Liu, Xu-dong; Mo, Xiumei; Fan, Cunyi

    2016-01-01

    Cartilage defects cause joint pain and loss of mobility. It is crucial to induce the chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by both biological and structural signals in cartilage tissue engineering. Sponge-like scaffolds fabricated using native cartilage extracellular matrix components can induce the BMSC differentiation by biological signals and limited structural signals. In this study, an oriented poly(L-lactic acid)-co-poly(ε-caprolactone) P(LLA-CL)/collagen type I (Col-I) nanofiber yarn mesh, fabricated by dynamic liquid electrospinning served as a skeleton for a freeze-dried Col-I/hyaluronate (HA) chondral phase (SPONGE) containing both structural and biological signals to guide BMSC chondrogenic differentiation. In vitro results show that the Yarn Col-I/HA hybrid scaffold (Yarn-CH) promotes orientation, adhesion and proliferation of BMSCs better than SPONGE. Furthermore, BMSCs seeded on the Yarn-CH scaffold demonstrated a large increase in the glycosaminoglycan content and expression of collagen type II following a 21-day culture. - Highlights: • An oriented yarn was used as the skeleton of the sponge-like scaffold. • Both structural and biological signals were given for BMSC chondrogenic differentiation. • Yarn-CH promotes orientation and chondrogenesis differentiation of BMSCs. • Yarn-CH reproduces the superficial zone of the cartilage.

  7. Chondrogenic Effect of Intra-articular Hypertonic-Dextrose (Prolotherapy) in Severe Knee Osteoarthritis.

    Science.gov (United States)

    Topol, Gastón Andrés; Podesta, Leandro Ariel; Reeves, Kenneth Dean; Giraldo, Marcia Mallma; Johnson, Lanny L; Grasso, Raul; Jamín, Alexis; Clark, Tom; Rabago, David

    2016-11-01

    Dextrose injection is reported to improve knee osteoarthritis (KOA)-related clinical outcomes, but its effect on articular cartilage is unknown. A chondrogenic effect of dextrose injection has been proposed. To assess biological and clinical effects of intra-articular hypertonic dextrose injections (prolotherapy) in painful KOA. Case series with blinded arthroscopic evaluation before and after treatment. Physical medicine and day surgery practice. Symptomatic KOA for at least 6 months, arthroscopy-confirmed medial compartment exposed subchondral bone, and temporary pain relief with intra-articular lidocaine injection. Four to 6 monthly 10-mL intra-articular injections with 12.5% dextrose. Visual cartilage growth assessment of 9 standardized medial condyle zones in each of 6 participants by 3 arthroscopy readers masked to pre-/postinjection status (total 54 zones evaluated per reader); biopsy of a cartilage growth area posttreatment, evaluated using hematoxylin and eosin and Safranin-O stains, quantitative polarized light microscopy, and immunohistologic cartilage typing; self-reported knee specific quality of life using the Western Ontario McMaster University Osteoarthritis Index (WOMAC, 0-100 points). Six participants (1 female and 5 male) with median age of 71 years, WOMAC composite score of 57.5 points, and a 9-year pain duration received a median of 6 dextrose injections and follow-up arthroscopy at 7.75 months (range 4.5-9.5 months). In 19 of 54 zone comparisons, all 3 readers agreed that the posttreatment zone showed cartilage growth compared with the pretreatment zone. Biopsy specimens showed metabolically active cartilage with variable cellular organization, fiber parallelism, and cartilage typing patterns consistent with fibro- and hyaline-like cartilage. Compared with baseline status, the median WOMAC score improved 13 points (P = .013). Self-limited soreness after methylene blue instillation was noted. Positive clinical and chondrogenic effects were seen

  8. 3D model of amphioxus steroid receptor complexed with estradiol

    Energy Technology Data Exchange (ETDEWEB)

    Baker, Michael E., E-mail: mbaker@ucsd.edu [Department of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0693 (United States); Chang, David J. [Department of Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0693 (United States)

    2009-08-28

    The origins of signaling by vertebrate steroids are not fully understood. An important advance was the report that an estrogen-binding steroid receptor [SR] is present in amphioxus, a basal chordate with a similar body plan as vertebrates. To investigate the evolution of estrogen-binding to steroid receptors, we constructed a 3D model of amphioxus SR complexed with estradiol. This 3D model indicates that although the SR is activated by estradiol, some interactions between estradiol and human ER{alpha} are not conserved in the SR, which can explain the low affinity of estradiol for the SR. These differences between the SR and ER{alpha} in the steroid-binding domain are sufficient to suggest that another steroid is the physiological regulator of the SR. The 3D model predicts that mutation of Glu-346 to Gln will increase the affinity of testosterone for amphioxus SR and elucidate the evolution of steroid-binding to nuclear receptors.

  9. Catabolic factors and osteoarthritis-conditioned medium inhibit chondrogenesis of human mesenchymal stem cells.

    Science.gov (United States)

    Heldens, Genoveva T H; Blaney Davidson, Esmeralda N; Vitters, Elly L; Schreurs, B Willem; Piek, Ester; van den Berg, Wim B; van der Kraan, Peter M

    2012-01-01

    Articular cartilage has a very limited intrinsic repair capacity leading to progressive joint damage. Therapies involving tissue engineering depend on chondrogenic differentiation of progenitor cells. This chondrogenic differentiation will have to survive in a diseased joint. We postulate that catabolic factors in this environment inhibit chondrogenesis of progenitor cells. We investigated the effect of a catabolic environment on chondrogenesis in pellet cultures of human mesenchymal stem cells (hMSCs). We exposed chondrogenically differentiated hMSC pellets, to interleukin (IL)-1α, tumor necrosis factor (TNF)-α or conditioned medium derived from osteoarthritic synovium (CM-OAS). IL-1α and TNF-α in CM-OAS were blocked with IL-1Ra or Enbrel, respectively. Chondrogenesis was determined by chondrogenic markers collagen type II, aggrecan, and the hypertrophy marker collagen type X on mRNA. Proteoglycan deposition was analyzed by safranin o staining on histology. IL-1α and TNF-α dose-dependently inhibited chondrogenesis when added at onset or during progression of differentiation, IL-1α being more potent than TNF-α. CM-OAS inhibited chondrogenesis on mRNA and protein level but varied in extent between patients. Inhibition of IL-1α partially overcame the inhibitory effect of the CM-OAS on chondrogenesis whereas the TNF-α contribution was negligible. We show that hMSC chondrogenesis is blocked by either IL-1α or TNF-α alone, but that there are additional factors present in CM-OAS that contribute to inhibition of chondrogenesis, demonstrating that catabolic factors present in OA joints inhibit chondrogenesis, thereby impairing successful tissue engineering.

  10. Stimulation of estradiol biosynthesis by tributyltin in rat hippocampal slices.

    Science.gov (United States)

    Munetsuna, Eiji; Hattori, Minoru; Yamazaki, Takeshi

    2014-01-01

    Hippocampal functions are influenced by steroid hormones, such as testosterone and estradiol. It has been demonstrated that hippocampus-derived steroid hormones play important roles in neuronal protection and synapse formation. Our research groups have demonstrated that estradiol is de novo synthesized in the rat hippocampus. However, the mechanism(s) regulating this synthesis remains unclear. It has been reported that tributyltin, an environmental pollutant, binds to the retinoid X receptor (RXR) and modifies estrogen synthesis in human granulosa-like tumor cells. This compound can penetrate the blood brain barrier, and tends to accumulate in the brain. Based on these facts, we hypothesized that tributyltin could influence the hippocampal estradiol synthesis. A concentration of 0.1 μM tributyltin induced an increase in the mRNA content of P450(17α) and P450arom in hippocampal slices, as determined using real-time PCR. The transcript levels of other steroidogenic enzymes and a steroidogenic acute regulatory protein were not affected. The estradiol level in rat hippocampal slices was subsequently determined using a radioimmunoassay. We found that the estradiol synthesis was stimulated by ∼2-fold following a 48-h treatment with 0.1 μM tributyltin, and this was accompanied by transcriptional activation of P450(17α) and P450arom. Tributyltin stimulated de novo hippocampal estradiol synthesis by modifying the transcription of specific steroidogenic enzymes.

  11. Chondrogenic potential of human mesenchymal stem cells and expression of Slug transcription factor.

    Science.gov (United States)

    Brini, Anna T; Niada, Stefania; Lambertini, Elisabetta; Torreggiani, Elena; Arrigoni, Elena; Lisignoli, Gina; Piva, Roberta

    2015-06-01

    The scientific literature rarely reports experimental failures or inconsistent outcomes in the induction of cell differentiation; however, researchers commonly experience poor or unsuccessful responses to differentiating agents when culturing stem cells. One way of investigating the underlying reasons for such responses is to look at the basal expression levels of specific genes in multipotent stem cells before the induction of differentiation. In addition to shedding light on the complex properties of stem cells and the molecular modulation of differentiation pathways, this strategy can also lead to the development of important time- and money-saving tools that aid the efficient selection of cellular specimens--in this case, stem cells that are more prone to differentiate towards specific lineages and are therefore more suitable for cell-based therapeutic protocols in regenerative medicine. To address this latter aspect, this study focused on understanding the reasons why some human mesenchymal stem cell (hMSC) samples are less efficient at differentiating towards chondrogenesis. This study shows that analysis of the basal expression levels of Slug, a negative regulator of chondrogenesis in hMSC, provides a rapid and simple tool for distinguishing stem cell samples with the potential to form a cartilage-like matrix, and that are therefore suitable for cartilage tissue engineering. It is shown that high basal levels of Slug prevent the chondrogenic differentiation of hMSCs, even in the presence of transforming growth factor-β and elevated levels of Sox9. Copyright © 2013 John Wiley & Sons, Ltd.

  12. The effects of 17β-estradiol and a selective estrogen receptor modulator, bazedoxifene, on ovarian carcinogenesis.

    Science.gov (United States)

    Romero, Iris L; Lee, WooSeok; Mitra, Anirban K; Gordon, Ilyssa O; Zhao, Yan; Leonhardt, Payton; Penicka, Carla V; Mui, Keeley L; Krausz, Thomas N; Greene, Geoffrey L; Lengyel, Ernst

    2012-01-01

    To test if estrogen promotes carcinogenesis in vitro and in a genetic mouse model of ovarian cancer and whether its effects can be inhibited by a novel selective estrogen receptor modulator (SERM), bazedoxifene. Bazedoxifene was synthesized and it was confirmed that the drug abrogated the uterine stimulatory effect of 17β-estradiol in mice. To determine if hormones alter tumorigenesis in vivo LSL-K-ras(G12D/+)Pten(loxP/loxP) mice were treated with vehicle control, 17β-estradiol or bazedoxifene. Hormone receptor status of a cell line established from LSL-K-ras(G12D/+)Pten(loxP/loxP) mouse ovarian tumors was characterized using Western blotting and immunohistochemistry. The cell line was treated with hormones and invasion assays were performed using Boyden chambers and proliferation was assessed using MTT assays. In vitro 17β-estradiol increased both the invasion and proliferation of ovarian cancer cells and bazedoxifene reversed these effects. However, in the genetic mouse model neither treatment with 17β-estradiol nor bazedoxifene changed mean tumor burden when compared to treatment with placebo. The mice in all treatment groups had similar tumor incidence, metastatic nodules and ascites. While 17β-estradiol increases the invasion and proliferation of ovarian cancer cells, these effects do not translate into increased tumor burden in a genetic mouse model of endometrioid ovarian cancer. Likewise, while the SERM reversed the detrimental effects of estrogen in vitro, there was no change in tumor burden in mice treated with bazedoxifene. These findings demonstrate the complex interplay between hormones and ovarian carcinogenesis. Copyright © 2011 Elsevier Inc. All rights reserved.

  13. Estradiol increases choice of cocaine over food in male rats.

    Science.gov (United States)

    Bagley, Jared R; Adams, Julia; Bozadjian, Rachel V; Bubalo, Lana; Ploense, Kyle L; Kippin, Tod E

    2017-10-19

    Estradiol modulates the rewarding and reinforcing properties of cocaine in females, including an increase in selection of cocaine over alternative reinforcers. However, the effects of estradiol on male cocaine self-administration behavior are less studied despite equivalent levels of estradiol in the brains of adult males and females, estradiol effects on motivated behaviors in males that share underlying neural substrates with cocaine reinforcement as well as expression of estrogen receptors in the male brain. Therefore, we sought to characterize the effects of estradiol in males on choice between concurrently-available cocaine and food reinforcement as well as responding for cocaine or food in isolation. Male castrated rats (n=46) were treated daily with estradiol benzoate (EB) (5μg/0.1, S.C.) or vehicle (peanut oil) throughout operant acquisition of cocaine (1mg/kg, IV; FI20 sec) and food (3×45mg; FI20 sec) responding, choice during concurrent access and cocaine and food reinforcement under progressive ratio (PR) schedules. EB increased cocaine choice, both in terms of percent of trials on which cocaine was selected and the proportion of rats exhibiting a cocaine preference as well as increased cocaine, but not food, intake under PR. Additionally, within the EB treated group, cocaine-preferring rats exhibited enhanced acquisition of cocaine, but not food, reinforcement whereas no acquisition differences were observed across preferences in the vehicle treated group. These findings demonstrate that estradiol increases cocaine choice in males similarly to what is observed in females. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Plasma concentrations of estradiol and testosterone, gonadal aromatase activity and ultrastructure of the testis in Xenopus laevis exposed to estradiol or atrazine

    International Nuclear Information System (INIS)

    Hecker, Markus; Kim, Wan Jong; Park, June-Woo; Murphy, Margaret B.; Villeneuve, Daniel; Coady, Katherine K.; Jones, Paul D.; Solomon, Keith R.; Kraak, Glen van der; Carr, James A.; Smith, Ernest E.; Preez, Louis du; Kendall, Ronald J.; Giesy, John P.

    2005-01-01

    The ultrastructure of testicular cells of adult male African clawed frogs (Xenopus laevis) exposed to either estradiol (0.1 μg/L) or 2-chloro-4-ethylamino-6-isopropyl-amino-s-triazine (atrazine; 10 or 100 μg/L) was examined by electron microscopy and compared to plasma concentrations of the steroid hormones, testosterone (T) and estradiol (E2), testicular aromatase activity and gonad growth expressed as the gonado-somatic index (GSI). Exposure to E2 caused significant changes both at the sub-cellular and biochemical levels. Exposure to E2 resulted in significantly fewer sperm cells, inhibition of meiotic division of germ cells, more lipid droplets that are storage compartments for the sex steroid hormone precursor cholesterol, and lesser plasma T concentrations. Although not statistically significant, frogs exposed to E2 had slightly smaller GSI values. These results may be indicative of an inhibition of gonad growth and disrupted germ cell development by E2. Concentrations of E2 in plasma were greater in frogs exposed to E2 in water. Exposure to neither concentration of atrazine caused effects on germ cell development, testicular aromatase activity or plasma hormone concentrations. These results suggest that atrazine does not affect testicular function. In contrast, exposure of male X. laevis to E2 led to sub-cellular events that are indicative of disruption of testicular development, and demasculinization processes (decrease of androgen hormone titers). These results indicate that atrazine does not cause responses that are similar to those caused by exposure to E2

  15. Estradiol Protects Proopiomelanocortin Neurons Against Insulin Resistance.

    Science.gov (United States)

    Qiu, Jian; Bosch, Martha A; Meza, Cecilia; Navarro, Uyen-Vy; Nestor, Casey C; Wagner, Edward J; Rønnekleiv, Oline K; Kelly, Martin J

    2018-02-01

    Insulin resistance is at the core of the metabolic syndrome, and men exhibit a higher incidence of metabolic syndrome than women in early adult life, but this sex advantage diminishes sharply when women reach the postmenopausal state. Because 17β-estradiol (E2) augments the excitability of the anorexigenic proopiomelanocortin (POMC) neurons, we investigated the neuroprotective effects of E2 against insulin resistance in POMC neurons from diet-induced obese (DIO) female and male mice. The efficacy of insulin to activate canonical transient receptor potential 5 (TRPC5) channels and depolarize POMC neurons was significantly reduced in DIO male mice but not in DIO female mice. However, the insulin response in POMC neurons was abrogated in ovariectomized DIO females but restored with E2 replacement. E2 increased T-type calcium channel Cav3.1 messenger RNA (mRNA) expression and whole-cell currents but downregulated stromal-interaction molecule 1 mRNA, which rendered POMC neurons more excitable and responsive to insulin-mediated TRPC5 channel activation. Moreover, E2 prevented the increase in suppressor of cytokine signaling-3 mRNA expression with DIO as seen in DIO males. As proof of principle, insulin [intracerebroventricular injection into the third ventricle (ICV)] decreased food intake and increased metabolism in female but not male guinea pigs fed a high-fat diet. The uncoupling of the insulin receptor from its downstream effector system was corroborated by the reduced expression of phosphorylated protein kinase B in the arcuate nucleus of male but not female guinea pigs following insulin. Therefore, E2 protects female POMC neurons from insulin resistance by enhancing POMC neuronal excitability and the coupling of insulin receptor to TRPC5 channel activation. Copyright © 2018 Endocrine Society.

  16. Significance of soluble growth factors in the chondrogenic response of human umbilical cord matrix stem cells in a porous three dimensional scaffold

    Directory of Open Access Journals (Sweden)

    RS Nirmal

    2013-11-01

    Full Text Available Stem cell based tissue engineering has emerged as a promising strategy for articular cartilage regeneration. Foetal derived mesenchymal stem cells (MSCs with their ease of availability, pluripotency and high expansion potential have been demonstrated to be an attractive cell source over adult MSCs. However, there is a need for optimisation of chondrogenic signals to direct the differentiation of these multipotent MSCs to chondrogenic lineage. In this study we have demonstrated the in vitro chondrogenesis of human umbilical cord matrix MSCs in three dimensional PVA-PCL (polyvinyl alcohol-polycaprolactone scaffolds in the presence of the individual growth factors TGFβ1, TGFβ3, IGF, BMP2 and their combination with BMP2. Gene expression, histology and immunohistology were evaluated after 28 d culture. The induced cells showed the feature of chondrocytes in their morphology and expression of typical chondrogenic extracellular matrix molecules. Moreover, the real-time PCR assay has shown the expression of gene markers of chondrogenesis, SOX9, collagen type II and aggrecan. The expression of collagen type I and collagen type X was also evaluated. This study has demonstrated the successful chondrogenic induction of human umbilical cord MSCs in 3D scaffolds. Interestingly, the growth factor combination of TGF-β3 and BMP-2 was found to be more effective for chondrogenesis as shown by the real-time PCR studies. The findings of this study suggest the importance of using growth factor combinations for successful chondrogenic differentiation of umbilical cord MSCs.

  17. Improved functional assessment of osteoarthritic knee joint after chondrogenically induced cell treatment.

    Science.gov (United States)

    Ude, C C; Ng, M H; Chen, C H; Htwe, O; Amaramalar, N S; Hassan, S; Djordjevic, I; Rani, R A; Ahmad, J; Yahya, N M; Saim, A B; Idrus, R B Hj

    2015-08-01

    Our previous studies on osteoarthritis (OA) revealed positive outcome after chondrogenically induced cells treatment. Presently, the functional improvements of these treated OA knee joints were quantified followed by evaluation of the mechanical properties of the engineered cartilages. Baseline electromyogram (EMGs) were conducted at week 0 (pre-OA), on the locomotory muscles of nine un-castrated male sheep (Siamese long tail cross) divided into controls, adipose-derived stem cells (ADSCs) and bone marrow stem cells (BMSCs), before OA inductions. Subsequent recordings were performed at week 7 and week 31 which were post-OA and post-treatments. Afterwards, the compression tests of the regenerated cartilage were performed. Post-treatment EMG analysis revealed that the control sheep retained significant reductions in amplitudes at the right medial gluteus, vastus lateralis and bicep femoris, whereas BMSCs and ADSCs samples had no further significant reductions (P < 0.05). Grossly and histologically, the treated knee joints demonstrated the presence of regenerated neo cartilages evidenced by the fluorescence of PKH26 tracker. Based on the International Cartilage Repair Society scores (ICRS), they had significantly lower grades than the controls (P < 0.05). The compression moduli of the native cartilages and the engineered cartilages differed significantly at the tibia plateau, patella femoral groove and the patella; whereas at the medial femoral condyle, they had similar moduli of 0.69 MPa and 0.40-0.64 MPa respectively. Their compression strengths at all four regions were within ±10 MPa. The tissue engineered cartilages provided evidence of functional recoveries associated to the structural regenerations, and their mechanical properties were comparable with the native cartilage. Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  18. Creating an Animal Model of Tendinopathy by Inducing Chondrogenic Differentiation with Kartogenin.

    Science.gov (United States)

    Yuan, Ting; Zhang, Jianying; Zhao, Guangyi; Zhou, Yiqin; Zhang, Chang-Qing; Wang, James H-C

    2016-01-01

    Previous animal studies have shown that long term rat treadmill running induces over-use tendinopathy, which manifests as proteoglycan accumulation and chondrocytes-like cells within the affected tendons. Creating this animal model of tendinopathy by long term treadmill running is however time-consuming, costly and may vary among animals. In this study, we used a new approach to develop an animal model of tendinopathy using kartogenin (KGN), a bio-compound that can stimulate endogenous stem/progenitor cells to differentiate into chondrocytes. KGN-beads were fabricated and implanted into rat Achilles tendons. Five weeks after implantation, chondrocytes and proteoglycan accumulation were found at the KGN implanted site. Vascularity as well as disorganization in collagen fibers were also present in the same site along with increased expression of the chondrocyte specific marker, collagen type II (Col. II). In vitro studies confirmed that KGN was released continuously from KGN-alginate in vivo beads and induced chondrogenic differentiation of tendon stem/progenitor cells (TSCs) suggesting that chondrogenesis after KGN-bead implantation into the rat tendons is likely due to the aberrant differentiation of TSCs into chondrocytes. Taken together, our results showed that KGN-alginate beads can be used to create a rat model of tendinopathy, which, at least in part, reproduces the features of over-use tendinopathy model created by long term treadmill running. This model is mechanistic (stem cell differentiation), highly reproducible and precise in creating localized tendinopathic lesions. It is expected that this model will be useful to evaluate the effects of various topical treatments such as NSAIDs and platelet-rich plasma (PRP) for the treatment of tendinopathy.

  19. Dydrogesterone does not reverse the cardiovascular benefits of percutaneous estradiol.

    Science.gov (United States)

    Kuba, V M; Teixeira, M A M; Meirelles, R M R; Assumpção, C R L; Costa, O S

    2013-02-01

    To evaluate the influence of dydrogesterone on estimated cardiovascular risk of users of hormone replacement therapy (HRT) (with percutaneous 17β-estradiol in monotherapy and in combination with dydrogesterone) and HRT non-users through the Framingham score tool for a period of 2 years. Framingham scores were calculated from the medical records of patients treated for at least 2 years with 17β-estradiol alone or in combination with dydrogesterone, along with HRT non-users, through the analysis of patient medical records, followed for at least 2 years at Instituto Estadual de Diabetes e Endocrinologia Luiz Capriglione. Improvements in lipid profile, glucose and blood pressure levels, which reduced the estimated cardiovascular risk, were observed in the 17β-estradiol group. Similar changes were observed in the users of 17β-estradiol + dydrogesterone, suggesting that this progestogen does not attenuate the effects caused by 17β-estradiol. Both HRT groups showed a reduction in their Framingham score. In contrast to data from other HRT investigations on cardiovascular risk, these formulations proved to be safe, even in the first year of use.

  20. Depletion of histone demethylase KDM2A enhanced the adipogenic and chondrogenic differentiation potentials of stem cells from apical papilla

    Energy Technology Data Exchange (ETDEWEB)

    Dong, Rui [Laboratory of Molecular Signaling and Stem Cells Therapy, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School of Stomatology, Beijing 100050 (China); Yao, Rui [Department of Pediatrics, Stomatological Hospital of Nankai University, Tianjin 300041 (China); Du, Juan [Laboratory of Molecular Signaling and Stem Cells Therapy, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School of Stomatology, Beijing 100050 (China); Wang, Songlin [Molecular Laboratory for Gene Therapy and Tooth Regeneration, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School of Stomatology, Beijing 100050 (China); Department of Biochemistry and Molecular Biology, Capital Medical University School of Basic Medical Sciences, Beijing 100069 (China); Fan, Zhipeng, E-mail: zpfan@ccmu.edu.cn [Laboratory of Molecular Signaling and Stem Cells Therapy, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School of Stomatology, Beijing 100050 (China)

    2013-11-01

    Mesenchymal stem cells (MSCs) are a reliable resource for tissue regeneration, but the molecular mechanism underlying directed differentiation remains unclear; this has restricted potential MSC applications. The histone demethylase, lysine (K)-specific demethylase 2A (KDM2A), is evolutionarily conserved and ubiquitously expressed members of the JmjC-domain-containing histone demethylase family. A previous study determined that KDM2A can regulate the cell proliferation and osteo/dentinogenic differentiation of MSCs. It is not known whether KDM2A is involved in the other cell lineages differentiation of MSCs. Here, we show that depletion of KDM2A by short hairpin RNAs can enhance adipogenic and chondrogenic differentiation potentials in human stem cells from apical papilla (SCAPs). We found that the stemness-related genes, SOX2, and the embryonic stem cell master transcription factor, NANOG were significantly increased after silence of KDM2A in SCAPs. Moreover, we found that knock-down of the KDM2A co-factor, BCOR also up-regulated the mRNA levels of SOX2 and NANOG. Furthermore, Chromatin immunoprecipitation assays demonstrate that silence of KDM2A increased the histone H3 Lysine 4 (H3K4) trimethylation in the SOX2 and NANOG locus and regulates its expression. In conclusion, our results suggested that depletion of KDM2A enhanced the adipogenic and chondrogenic differentiation potentials of SCAPs by up-regulated SOX2 and NANOG, BCOR also involved in this regulation as co-factor, and provided useful information to understand the molecular mechanism underlying directed differentiation in MSCs. - Highlights: • Depletion of KDM2A enhances adipogenic/chondrogenic differentiation in SCAPs. • Depletion of KDM2A enhances the differentiation of SCAPs by activate SOX2 and NANOG. • Silence of KDM2A increases histone H3 Lysine 4 trimethylation in SOX2 and NANOG. • BCOR is co-factor of KDM2A involved in the differentiation regulation.

  1. Multilayered dense collagen-silk fibroin hybrid: a platform for mesenchymal stem cell differentiation towards chondrogenic and osteogenic lineages.

    Science.gov (United States)

    Ghezzi, Chiara E; Marelli, Benedetto; Donelli, Ilaria; Alessandrino, Antonio; Freddi, Giuliano; Nazhat, Showan N

    2017-07-01

    Type I collagen is a major structural and functional protein in connective tissues. However, collagen gels exhibit unstable geometrical properties, arising from extensive cell-mediated contraction. In an effort to stabilize collagen-based hydrogels, plastic compression was used to hybridize dense collagen (DC) with electrospun silk fibroin (SF) mats, generating multilayered DC-SF-DC constructs. Seeded mesenchymal stem cell (MSC)-mediated DC-SF-DC contraction, as well as growth and differentiation under chondrogenic and osteogenic supplements, were compared to those seeded in DC and on SF alone. The incorporation of SF within DC prevented extensive cell-mediated collagen gel contraction. The effect of the multilayered hybrid on MSC remodelling capacity was also evident at the transcription level, where the expression of matrix metalloproteinases and their inhibitor (MMP1, MMP2, MMP3, MMP13 and Timp1) by MSCs within DC-SF-DC were comparable to those on SF and significantly downregulated in comparison to DC, except for Timp1. Chondrogenic supplements stimulated extracellular matrix production within the construct, stabilizing its multilayered structure and promoting MSC chondrogenic differentiation, as indicated by the upregulation of the genes Col2a1 and Agg and the production of collagen type II. In osteogenic medium there was an upregulation in ALP and OP along with the presence of an apatitic phase, indicating MSC osteoblastic differentiation and matrix mineralization. In sum, these results have implications on the modulation of three-dimensional collagen-based gel structural stability and on the stimulation and maintenance of the MSC committed phenotype inherent to the in vitro formation of chondral tissue and bone, as well as on potential multilayered complex tissues. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  2. TiO2 coating promotes human mesenchymal stem cell proliferation without the loss of their capacity for chondrogenic differentiation

    International Nuclear Information System (INIS)

    Kaitainen, Salla; Lappalainen, Reijo; Mähönen, Anssi J; J Lammi, Mikko; Qu, Chengjuan; Kröger, Heikki

    2013-01-01

    Human mesenchymal stem cells (hMSCs) are used in applications, which may require a large amount of cells; therefore, efficient expansion of the cells is desired. We studied whether TiO 2 coating on plastic cell culture dishes could promote proliferation of hMSCs without adverse effects in chondrogenic differentiation. TiO 2 -films were deposited on polystyrene dishes and glass coverslips using an ultrashort pulsed laser deposition technique. Human MSCs from three donors were expanded on them until 95% confluence, and the cells were evaluated by morphology, immunocytochemistry and quantitative RT-PCR (qRT-PCR). The chondrogenic differentiation in pellets was performed after cultivation on TiO 2 -coated dishes. Chondrogenesis was evaluated by histological staining of proteoglycans and type II collagen, and qRT-PCR. Human MSC-associated markers STRO-1, CD44, CD90 and CD146 did not change after expansion on TiO 2 -coated coverslips. However, the cell number after a 48h-culture period was significantly higher on TiO 2 -coated culture dishes. Importantly, TiO 2 coating caused no significant differences in the proteoglycan and type II collagen staining of the pellets, or the expression of chondrocyte-specific genes in the chondrogenesis assay. Thus, the proliferation of hMSCs could be significantly increased when cultured on TiO 2 -coated dishes without weakening their chondrogenic differentiation capacity. The transparency of TiO 2 -films allows easy monitoring of the cell growth and morphology under a phase-contrast microscope. (paper)

  3. The effect of dexamethasone and triiodothyronine on terminal differentiation of primary bovine chondrocytes and chondrogenically differentiated mesenchymal stem cells.

    Science.gov (United States)

    Randau, Thomas M; Schildberg, Frank A; Alini, Mauro; Wimmer, Matthias D; Haddouti, El-Mustapha; Gravius, Sascha; Ito, Keita; Stoddart, Martin J

    2013-01-01

    The newly evolved field of regenerative medicine is offering solutions in the treatment of bone or cartilage loss and deficiency. Mesenchymal stem cells, as well as articular chondrocytes, are potential cells for the generation of bone or cartilage. The natural mechanism of bone formation is that of endochondral ossification, regulated, among other factors, through the hormones dexamethasone and triiodothyronine. We investigated the effects of these hormones on articular chondrocytes and chondrogenically differentiated mesenchymal stem cells, hypothesizing that these hormones would induce terminal differentiation, with chondrocytes and differentiated stem cells being similar in their response. Using a 3D-alginate cell culture model, bovine chondrocytes and chondrogenically differentiated stem cells were cultured in presence of triiodothyronine or dexamethasone, and cell proliferation and extracellular matrix production were investigated. Collagen mRNA expression was measured by real-time PCR. Col X mRNA and alkaline phosphatase were monitored as markers of terminal differentiation, a prerequisite of endochondral ossification. The alginate culture system worked well, both for the culture of chondrocytes and for the chondrogenic differentiation of mesenchymal stem cells. Dexamethasone led to an increase in glycosaminoglycan production. Triiodothyronine increased the total collagen production only in chondrocytes, where it also induced signs of terminal differentiation, increasing both collagen X mRNA and alkaline phosphatase activity. Dexamethasone induced terminal differentiation in the differentiated stem cells. The immature articular chondrocytes used in this study seem to be able to undergo terminal differentiation, pointing to their possible role in the onset of degenerative osteoarthritis, as well as their potential for a cell source in bone tissue engineering. When chondrocyte-like cells, after their differentiation, can indeed be moved on towards terminal

  4. Depletion of histone demethylase KDM2A enhanced the adipogenic and chondrogenic differentiation potentials of stem cells from apical papilla

    International Nuclear Information System (INIS)

    Dong, Rui; Yao, Rui; Du, Juan; Wang, Songlin; Fan, Zhipeng

    2013-01-01

    Mesenchymal stem cells (MSCs) are a reliable resource for tissue regeneration, but the molecular mechanism underlying directed differentiation remains unclear; this has restricted potential MSC applications. The histone demethylase, lysine (K)-specific demethylase 2A (KDM2A), is evolutionarily conserved and ubiquitously expressed members of the JmjC-domain-containing histone demethylase family. A previous study determined that KDM2A can regulate the cell proliferation and osteo/dentinogenic differentiation of MSCs. It is not known whether KDM2A is involved in the other cell lineages differentiation of MSCs. Here, we show that depletion of KDM2A by short hairpin RNAs can enhance adipogenic and chondrogenic differentiation potentials in human stem cells from apical papilla (SCAPs). We found that the stemness-related genes, SOX2, and the embryonic stem cell master transcription factor, NANOG were significantly increased after silence of KDM2A in SCAPs. Moreover, we found that knock-down of the KDM2A co-factor, BCOR also up-regulated the mRNA levels of SOX2 and NANOG. Furthermore, Chromatin immunoprecipitation assays demonstrate that silence of KDM2A increased the histone H3 Lysine 4 (H3K4) trimethylation in the SOX2 and NANOG locus and regulates its expression. In conclusion, our results suggested that depletion of KDM2A enhanced the adipogenic and chondrogenic differentiation potentials of SCAPs by up-regulated SOX2 and NANOG, BCOR also involved in this regulation as co-factor, and provided useful information to understand the molecular mechanism underlying directed differentiation in MSCs. - Highlights: • Depletion of KDM2A enhances adipogenic/chondrogenic differentiation in SCAPs. • Depletion of KDM2A enhances the differentiation of SCAPs by activate SOX2 and NANOG. • Silence of KDM2A increases histone H3 Lysine 4 trimethylation in SOX2 and NANOG. • BCOR is co-factor of KDM2A involved in the differentiation regulation

  5. Effect on growth and cell cycle kinetics of estradiol and tamoxifen on MCF-7 human breast cancer cells grown in vitro and in nude mice

    DEFF Research Database (Denmark)

    Brünner, N; Bronzert, D; Vindeløv, L L

    1989-01-01

    The effects of estradiol and tamoxifen (TAM) on the estrogen-dependent human breast cancer cell line MCF-7 grown in vitro and in nude mice were compared. The effect on growth was determined by cell number in vitro and by tumor growth curves in nude mice. The effects on the cell cycle kinetics were...... determined by repeated flow cytometric DNA analyses in vitro and in vivo and by the technique of labeled mitosis in nude mouse-grown tumors. Under in vitro conditions, estradiol induced a pronounced increase in S-phase fraction and cell number. TAM inhibited growth of MCF-7 cells with a concomitant increase...... in the G1 phase from 60% to 75%. In nude mice, MCF-7 only formed tumors in estradiol-supplemented mice. No differences were observed in growth and cell kinetics between 0.1 and 1.0 mg of estradiol. Daily i.p. injections of TAM resulted in tumor growth inhibition with shrinkage of tumors. The flow...

  6. Incorporating pTGF-β1/calcium phosphate nanoparticles with fibronectin into 3-dimensional collagen/chitosan scaffolds: Efficient, sustained gene delivery to stem cells for chondrogenic differentiation

    Directory of Open Access Journals (Sweden)

    X Cao

    2012-02-01

    Full Text Available The objective of this study was to prepare a 3-dimensional nanoparticle gene delivery system (3D-NGDS based on collagen/chitosan scaffolds, in which plasmid transforming growth factor beta 1 (TGF-β1/calcium phosphate nanoparticles mixed with fibronectin (FN were used to transfect mesenchymal stem cells (MSCs. Scanning electron microscopy was used to characterise the microstructure of 3-dimensional collagen/chitosan scaffolds. An analysis performed to quantify the TGF-b1 concentrations in MSC cultures revealed that the MSCs transfected with the 3D-NGDS showed remarkably high levels of TGF-b1 over long periods, retaining a concentration of TGF-b1 of approximately 10 ng/mL within two weeks, with the highest level (12.6 ng/mL being observed on the 6th day. An immunohistochemistry analysis for collagen type II revealed that much higher production of collagen II from the 9th to 15th day was observed in the 3D-NGDS-transfected MSCs than that in MSCs transfected by the Lipofectamine 2000 method. The glycosaminoglycan content of the 3D-NGDS was comparable to those treated with TGF-β1 as well as TGF-β1 plus dexamethasone, and was significantly higher than those treated with free plasmid and Lipofectamine 2000. A remarkable type I collagen expression inhibition of the 3D-NGDS at day 21 was observed via ELISA. These results suggested that transfection with the 3D-NGDS could successfully induce MSC chondrogenic differentiation in vitro without dexamethasone. In summary, the 3D-NGDS could be developed into a promising alternative method to transfer exogenous nucleic acid to MSCs in clinical trials.

  7. Differential effects of BMP-2 and TGF-beta1 on chondrogenic differentiation of adipose derived stem cells

    DEFF Research Database (Denmark)

    Mehlhorn, A T; Niemeyer, P; Kaschte, K

    2007-01-01

    transcriptional regulation of Dlx-5, Msx-2 and Runx-2. MATERIALS AND METHODS: Encapsulated ASC were cultured for 14 days in medium containing TGF-beta1 and/or BMP-2. mRNA expression of the extracellular matrix molecules col2a1, cartilage oligomeric matrix protein, col10a1, alkaline phosphatase (AP......) and transcription factors Msx-2, Dlx-5 and Runx-2 was analysed. Release of glycosaminoglycans, collagen types II and X into the extracellular matrix was demonstrated. RESULTS: BMP-2 and TGF-beta1 induced a chondrogenic phenotype in ASC. Combined growth factor treatment had a synergistic effect on col10a1...

  8. Platelet-Rich Plasma Preparation Types Show Impact on Chondrogenic Differentiation, Migration, and Proliferation of Human Subchondral Mesenchymal Progenitor Cells.

    Science.gov (United States)

    Kreuz, Peter Cornelius; Krüger, Jan Philipp; Metzlaff, Sebastian; Freymann, Undine; Endres, Michaela; Pruss, Axel; Petersen, Wolf; Kaps, Christian

    2015-10-01

    To evaluate the chondrogenic potential of platelet concentrates on human subchondral mesenchymal progenitor cells (MPCs) as assessed by histomorphometric analysis of proteoglycans and type II collagen. Furthermore, the migratory and proliferative effect of platelet concentrates were assessed. Platelet-rich plasma (PRP) was prepared using preparation kits (Autologous Conditioned Plasma [ACP] Kit [Arthrex, Naples, FL]; Regen ACR-C Kit [Regen Lab, Le Mont-Sur-Lausanne, Switzerland]; and Dr.PRP Kit [Rmedica, Seoul, Republic of Korea]) by apheresis (PRP-A) and by centrifugation (PRP-C). In contrast to clinical application, freeze-and-thaw cycles were subsequently performed to activate platelets and to prevent medium coagulation by residual fibrinogen in vitro. MPCs were harvested from the cortico-spongious bone of femoral heads. Chondrogenic differentiation of MPCs was induced in high-density pellet cultures and evaluated by histochemical staining of typical cartilage matrix components. Migration of MPCs was assessed using a chemotaxis assay, and proliferation activity was measured by DNA content. MPCs cultured in the presence of 5% ACP, Regen, or Dr.PRP formed fibrous tissue, whereas MPCs stimulated with 5% PRP-A or PRP-C developed compact and dense cartilaginous tissue rich in type II collagen and proteoglycans. All platelet concentrates significantly (ACP, P = .00041; Regen, P = .00029; Dr.PRP, P = .00051; PRP-A, P platelet concentrates but one (Dr.PRP, P = .63) showed a proliferative effect on MPCs, as shown by significant increases (ACP, P = .027; Regen, P = .0029; PRP-A, P = .00021; and PRP-C, P = .00069) in DNA content. Platelet concentrates obtained by different preparation methods exhibit different potentials to stimulate chondrogenic differentiation, migration, and proliferation of MPCs. Platelet concentrates obtained by commercially available preparation kits failed to induce chondrogenic differentiation of MPCs, whereas highly standardized PRP

  9. Complete human serum maintains viability and chondrogenic potential of human synovial stem cells: suitable conditions for transplantation.

    Science.gov (United States)

    Mizuno, Mitsuru; Katano, Hisako; Otabe, Koji; Komori, Keiichiro; Kohno, Yuji; Fujii, Shizuka; Ozeki, Nobutake; Horie, Masafumi; Tsuji, Kunikazu; Koga, Hideyuki; Muneta, Takeshi; Sekiya, Ichiro

    2017-06-13

    In our clinical practice, we perform transplantations of autologous synovial mesenchymal stem cells (MSCs) for cartilage and meniscus regenerative medicine. One of the most important issues to ensuring clinical efficacy involves the transport of synovial MSCs from the processing facility to the clinic. Complete human serum (100% human serum) is an attractive candidate material in which to suspend synovial MSCs for their preservation during transport. The purpose of this study was to investigate whether complete human serum maintained MSC viability and chondrogenic potential and to examine the optimal temperature conditions for the preservation of human synovial MSCs. Human synovium was harvested from the knees of 14 donors with osteoarthritis during total knee arthroplasty. Passage 2 synovial MSCs were suspended at 2 million cells/100 μL in Ringer's solution or complete human serum at 4, 13, and 37 °C for 48 h. These cells were analyzed for live cell rates, cell surface marker expression, metabolic activity, proliferation, and adipogenic, calcification, and chondrogenic differentiation potentials before and after preservation. After preservation, synovial MSCs maintained higher live cell rates in human serum than in Ringer's solution at 4 and 13 °C. Synovial MSCs preserved in human serum at 4 and 13 °C also maintained high ratios of propidium iodide - and annexin V - cells. MSC surface marker expression was not altered in cells preserved at 4 and 13 °C. The metabolic activities of cells preserved in human serum at 4 and 13 °C was maintained, while significantly reduced in other conditions. Replated MSCs retained their proliferation ability when preserved in human serum at 4 and 13 °C. Adipogenesis and calcification potential could be observed in cells preserved in each condition, whereas chondrogenic potential was retained only in cells preserved in human serum at 4 and 13 °C. The viability and chondrogenic potential of synovial MSCs were

  10. β-Estradiol and ethinyl-estradiol contamination in the rivers of the Carpathian Basin.

    Science.gov (United States)

    Avar, Péter; Zrínyi, Zita; Maász, Gábor; Takátsy, Anikó; Lovas, Sándor; G-Tóth, László; Pirger, Zsolt

    2016-06-01

    17β-Estradiol (E2) and 17α-ethinyl estradiol (EE2), which are environmental estrogens, have been determined with LC-MS in freshwater. Their sensitive analysis needs derivatization and therefore is very hard to achieve in multiresidue screening. We analyzed samples from all the large and some small rivers (River Danube, Drava, Mur, Sava, Tisza, and Zala) of the Carpathian Basin and from Lake Balaton. Freshwater was extracted on solid phase and derivatized using dansyl chloride. Separation was performed on a Kinetex XB-C18 column. Detection was achieved with a benchtop orbitrap mass spectrometer using targeted MS analysis for quantification. Limits of quantification were 0.05 ng/L (MS1) and 0.1 ng/L (MS/MS) for E2, and 0.001 ng/L (MS1) and 0.2 ng/L (MS/MS) for EE2. River samples contained n.d.-5.2 ng/L E2 and n.d.-0.68 ng/L EE2. Average levels of E2 and EE2 were 0.61 and 0.084 ng/L, respectively, in rivers, water courses, and Lake Balaton together, but not counting city canal water. EE2 was less abundant, but it was still present in almost all of the samples. In beach water samples from Lake Balaton, we measured 0.076-0.233 E2 and n.d.-0.133 EE2. A relative high amount of EE2 was found in river Zala (0.68 ng/L) and in Hévíz-Páhoki canal (0.52 ng/L), which are both in the catchment area of Lake Balaton (Hungary).

  11. Estradiol stimulation of inositolphospholipid metabolism in human endometrial fibroblasts

    International Nuclear Information System (INIS)

    Iida, K.; Imai, A.; Tamaya, T.

    1989-01-01

    Stimulated inositolphospholipid turnover has been proposed to constitute a signal-transducing mechanism in many cell types. To determine the inositolphospholipid turnover during stimulation by 17 beta-estradiol, the turnover kinetics of phospholipids was investigated in human endometrial fibroblasts. In cells incubated with [ 32 P] phosphate for 1 h, estradiol rapidly and persisitently (for at least 30 min) enhanced the rate of 32 P-labeling of phosphatidic acid (PA). On the other hand, after a lag time of 5 min, 32 P-labeling of phosphatidylinositol (PI) was also increased also. These sequential 32 P-labeling of PA and PI demonstrated that inositolphospholipid turnover was stimulated in fibroblasts exposed to estradiol. The rapid estrogen-stimulated inositolphospholipid turnover may not be through the mechanism associated with classical action of estrogen

  12. Plasma and faecal testosterone and estradiol in chicken

    International Nuclear Information System (INIS)

    Mekchay, S.; Apichartsrungkoon, T.; Pongpiachan, P.

    1996-01-01

    Identification of sex in some kind of fowls can not be done by using their external appearances. Sex steroid hormone levels may be used as an indicator of sexual dimorphism in birds. The objective of this investigation was to measure plasma and faecal testosterone and estradiol concentrations in 8 male and 15 female chickens by using radioimmunoassay (RIA) technique. The relationship between plasma and faecal testosterone, and plasma and faecal estradiol are positively correlated. The correlation coefficients (r 2 ) between plasma and faecal steroids concentration were 0.621 (p<0.05) for testosterone and 0.692 (p<0.05) for estradiol. The average plasma and faecal sex steroid concentrations in male and female chickens were 10.05 ± 1.97 ng/ml and 511.50 ± 95.89 ng/g (for male testosterone), 24.85 ± 1.60 pg/ml and 49.65 ± 6.01 ng/g (for male estradiol), 0.79 ± 0.05 ng/ml and 134.20 ± 14.70 ng/ml (for female testosterone), 129.91 ± 19.30 pg/ml and 334.80 ± 15.62 ng/g (for female estradiol), respectively. Plasma and faecal testosterone and estradiol levels in male and female chickens are significant difference (p<0.01, p<0.01, p<0.001 and p<0.001 respectively). The results of this investigation suggested that plasma or faecal sex steroid concentrations can be used to discriminate sex of chicken which is show the possibility to use the plasma or faecal sex steroids for identification of sex in other bird species

  13. Dissipation of 17β-estradiol in composted poultry litter.

    Science.gov (United States)

    Hakk, Heldur; Sikora, Lawrence

    2011-01-01

    The excreted estrogen rate of all livestock in the United States is estimated at 134 kg d. The influence of manure treatment on the fate of estrogens is critical in deciding the recycling of over 300 million dry tons of livestock produced annually. The effects of two common manure management practices, heated composting and ambient temperature decomposition, on the fate of 17β-estradiol in poultry litter were determined. A mixture of poultry litter, wood chips, and straw was amended with [C]17β-estradiol and allowed to undergo decomposition with a laboratory-scale heated composter (HC) or room temperature incubation (RTI) for 24 d. Radiolabel in the finished products was fractionated into water-extractable, acetone-extractable, nonextractable, and mineralized fractions. Total 17β-estradiol radioactive residues in the HC and RTI ( = 2) treatments were not different ( > 0.05), except that statistically less 17β-estradiol was mineralized to CO during HC than RTI (1.1 vs. 10.0% for HC and RTI, respectively). Estrone was the major degradation product in extracts of HC and RTI treatments as determined by liquid chromatography/mass spectrometry analyses. The nonextractable residues indicated no quantitative differences among the humins between the treatments. An estimated 3% of the fortified estrogenicity remained after HC treatment, and 15% of the fortified estrogenicity remained after RTI treatment. If reduction of water-removable, biologically active 17β-estradiol is the treatment goal, then HC treatment would be slightly preferred over ambient temperature degradation. However, unmanaged, ambient temperature litter piles are less costly and time consuming for food animal producers and result in greater mineralization and similar immobilization of estradiol. by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Inc.

  14. Synthesis and evaluation of 7α-(3-[18F]fluoropropyl) estradiol

    International Nuclear Information System (INIS)

    Okamoto, Mayumi; Naka, Kyosuke; Kitagawa, Yuya; Ishiwata, Kiichi; Yoshimoto, Mitsuyoshi; Shimizu, Isao; Toyohara, Jun

    2015-01-01

    estradiol dose-dependently inhibited C3-7α-[ 18 F]FES uptake in the uterus and ovary. The in vivo IC 50 values of estradiol in the uterus and ovary were 34.4 and 38.5 nmol/kg, respectively. Furthermore, in vivo tumor uptake of C3-7α-[ 18 F]FES was significantly higher (unpaired t test with Welch’s correction; p = 0.015) in ER-positive T-47D tumors (2.3%ID/g ± 0.4%ID/g) than ER-negative MDA-MB-231 tumors (0.9%ID/g ± 0.1%ID/g). Conclusions: Although extensive metabolism was observed in rodents, C3-7α-[ 18 F]FES showed promising results for quantitative analysis of ER density in vivo. However, the selective uptake of C3-7α-[ 18 F]FES was lower than that of 16α-[ 18 F]FES. Further optimizations and structure–activity relationship studies of the C-7α-substituted estradiol are needed

  15. Effects of 17β-estradiol and progesterone on the production of adipokines in differentiating 3T3-L1 adipocytes: Role of Rho-kinase.

    Science.gov (United States)

    Pektaş, Mehtap; Kurt, Akif Hakan; Ün, İsmail; Tiftik, Rukiye Nalan; Büyükafşar, Kansu

    2015-04-01

    Effect of female sex hormones on the production/release of adipocyte-derived cytokines has been debatable. Furthermore, whether the cellular signaling triggered by these hormones involve Rho-kinase has not been investigated yet. Therefore, in this study, effects of 17β-estradiol and progesterone as well as the Rho-kinase inhibitor, Y-27632 on the level of adipokines such as resistin, adiponectin, leptin, TNF-α and IL-6 were investigated in 3T3-L1-derived adipocytes. Differentiation was induced in the post-confluent preadipocytes by the standard differentiation medium (Dulbecco's modified Eagle's medium with 10% fetal bovine serum together with the mixture of isobutylmethylxanthine, dexamethasone and insulin) in the presence of 17β-estradiol (10(-8)-10(-7)M), progesterone (10(-6)-10(-5)M), the Rho-kinase inhibitor, Y-27632 (10(-5)M) and their combination for 8days. Measurements of the adipokines were performed in the culturing medium by ELISA kits using specific monoclonal antibodies. 17β-estradiol elevated resistin but decreased adiponectin and IL-6 levels; however, it did not alter the concentration of leptin and TNF-α. Y-27632 pretreatment inhibited the rise of resistin and the fall of adiponectin by 17β-estradiol without any effects by its own. Progesterone did not change resistin, leptin and TNF-α level; however, it elevated adiponectin and decreased IL-6 production. Neither 17β-estradiol nor Y-27632 was able to antagonize the increase of adiponectin and the reduction of IL-6 levels by progesterone. While Y-27632 alone lowered IL-6 level, it increased leptin and TNF-α concentration without altering resistin and adiponectin. In conclusion, 17β-estradiol could modify adipokine production in 3T3-L1 adipocytes with the actions some of which involve Rho-kinase mediation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Peripheral blood aspirates overexpressing IGF-I via rAAV gene transfer undergo enhanced chondrogenic differentiation processes.

    Science.gov (United States)

    Frisch, Janina; Orth, Patrick; Rey-Rico, Ana; Venkatesan, Jagadeesh Kumar; Schmitt, Gertrud; Madry, Henning; Kohn, Dieter; Cucchiarini, Magali

    2017-11-01

    Implantation of peripheral blood aspirates induced towards chondrogenic differentiation upon genetic modification in sites of articular cartilage injury may represent a powerful strategy to enhance cartilage repair. Such a single-step approach may be less invasive than procedures based on the use of isolated or concentrated MSCs, simplifying translational protocols in patients. In this study, we provide evidence showing the feasibility of overexpressing the mitogenic and pro-anabolic insulin-like growth factor I (IGF-I) in human peripheral blood aspirates via rAAV-mediated gene transfer, leading to enhanced proliferative and chondrogenic differentiation (proteoglycans, type-II collagen, SOX9) activities in the samples relative to control (reporter rAAV-lacZ) treatment over extended periods of time (at least 21 days, the longest time-point evaluated). Interestingly, IGF-I gene transfer also triggered hypertrophic, osteo- and adipogenic differentiation processes in the aspirates, suggesting that careful regulation of IGF-I expression may be necessary to contain these events in vivo. Still, the current results demonstrate the potential of targeting human peripheral blood aspirates via therapeutic rAAV transduction as a novel, convenient tool to treat articular cartilage injuries. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  17. Effects of Serial Passage on the Characteristics and Chondrogenic Differentiation of Canine Umbilical Cord Matrix Derived Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    K. S. Lee

    2013-04-01

    Full Text Available Mesenchymal stem cells (MSCs are often known to have a therapeutic potential in the cell-mediated repair for fatal or incurable diseases. In this study, canine umbilical cord MSCs (cUC-MSCs were isolated from umbilical cord matrix (n = 3 and subjected to proliferative culture for 5 consecutive passages. The cells at each passage were characterized for multipotent MSC properties such as proliferation kinetics, expression patterns of MSC surface markers and self-renewal associated markers, and chondrogenic differentiation. In results, the proliferation of the cells as determined by the cumulative population doubling level was observed at its peak on passage 3 and stopped after passage 5, whereas cell doubling time dramatically increased after passage 4. Expression of MSC surface markers (CD44, CD54, CD61, CD80, CD90 and Flk-1, molecule (HMGA2 and pluripotent markers (sox2, nanog associated with self-renewal was negatively correlated with the number of passages. However, MSC surface marker (CD105 and pluripotent marker (Oct3/4 decreased with increasing the number of subpassage. cUC-MSCs at passage 1 to 5 underwent chondrogenesis under specific culture conditions, but percentage of chondrogenic differentiation decreased with increasing the number of subpassage. Collectively, the present study suggested that sequential subpassage could affect multipotent properties of cUC-MSCs and needs to be addressed before clinical applications.

  18. Chondrogenic potential of bone marrow–derived mesenchymal stem cells on a novel, auricular-shaped, nanocomposite scaffold

    Directory of Open Access Journals (Sweden)

    Kavi H Patel

    2013-12-01

    Full Text Available Reconstruction of the human auricle remains a challenge to plastic surgeons, and current approaches are not ideal. Tissue engineering provides a promising alternative. This study aims to evaluate the chondrogenic potential of bone marrow–derived mesenchymal stem cells on a novel, auricular-shaped polymer. The proposed polyhedral oligomeric silsesquioxane-modified poly(hexanolactone/carbonateurethane/urea nanocomposite polymer has already been transplanted in patients as the world’s first synthetic trachea, tear duct and vascular bypass graft. The nanocomposite scaffold was fabricated via a coagulation/salt-leaching method and shaped into an auricle. Adult bone marrow–derived mesenchymal stem cells were isolated, cultured and seeded onto the scaffold. On day 21, samples were sent for scanning electron microscopy, histology and immunofluorescence to assess for neocartilage formation. Cell viability assay confirmed cytocompatability and normal patterns of cellular growth at 7, 14 and 21 days after culture. This study demonstrates the potential of a novel polyhedral oligomeric silsesquioxane-modified poly(hexanolactone/carbonateurethane/urea scaffold for culturing bone marrow–derived mesenchymal stem cells in chondrogenic medium to produce an auricular-shaped construct. This is supported by scanning electron microscopy, histological and immunofluorescence analysis revealing markers of chondrogenesis including collagen type II, SOX-9, glycosaminoglycan and elastin. To the best of our knowledge, this is the first report of stem cell application on an auricular-shaped scaffold for tissue engineering purposes. Although many obstacles remain in producing a functional auricle, this is a promising step forward.

  19. Directing chondrogenic differentiation of mesenchymal stem cells with a solid-supported chitosan thermogel for cartilage tissue engineering

    International Nuclear Information System (INIS)

    Huang, Hongjie; Zhang, Xin; Hu, Xiaoqing; Dai, Linghui; Zhu, Jingxian; Man, Zhentao; Ao, Yingfang; Chen, Haifeng; Zhou, Chunyan

    2014-01-01

    Hydrogels are attractive for cartilage tissue engineering because of their high plasticity and similarity with the native cartilage matrix. However, one critical drawback of hydrogels for osteochondral repair is their inadequate mechanical strength. To address this limitation, we constructed a solid-supported thermogel comprising a chitosan hydrogel system and demineralized bone matrix. Scanning electron microscopy, the equilibrium scanning ratio, the biodegradation rate, biomechanical tests, biochemical assays, metabolic activity tests, immunostaining and cartilage-specific gene expression analysis were used to evaluate the solid-supported thermogel. Compared with pure hydrogel or demineralized matrix, the hybrid biomaterial showed superior porosity, equilibrium swelling and degradation rate. The hybrid scaffolds exhibited an increased mechanical strength: 75% and 30% higher compared with pure hydrogels and demineralized matrix, respectively. After three days culture, bone-derived mesenchymal stem cells (BMSCs) maintained viability above 90% in all three materials; however, the cell retention of the hybrid scaffolds was more efficient and uniform than the other materials. Matrix production and chondrogenic differentiation of BMSCs in the hybrid scaffolds were superior to its precursors, based on glycosaminoglycan quantification and hyaline cartilage marker expression after three weeks in culture. Its easy preparation, favourable biophysical properties and chondrogenic capacity indicated that this solid-supported thermogel could be an attractive biomaterial framework for cartilage tissue engineering. (paper)

  20. Long-term dynamic loading improves the mechanical properties of chondrogenic mesenchymal stem cell-laden hydrogel

    Directory of Open Access Journals (Sweden)

    AH Huang

    2010-02-01

    Full Text Available Mesenchymal stem cells (MSCs are an attractive cell source for cartilage tissue engineering given their ability to undergo chondrogenesis in 3D culture systems. Mechanical forces play an important role in regulating both cartilage development and MSC chondrogenic gene expression, however, mechanical stimulation has yet to enhance the mechanical properties of engineered constructs. In this study, we applied long-term dynamic compression to MSC-seeded constructs and assessed whether varying pre-culture duration, loading regimens and inclusion of TGF-beta3 during loading would influence functional outcomes and these phenotypic transitions. Loading initiated before chondrogenesis decreased functional maturation, although chondrogenic gene expression increased. In contrast, loading initiated after chondrogenesis and matrix elaboration further improved the mechanical properties of MSC-based constructs, but only when TGF-beta3 levels were maintained and under specific loading parameters. Although matrix quantity was not affected by dynamic compression, matrix distribution, assessed histologically and by FT-IRIS analysis, was significantly improved on the micro- (pericellular and macro- (construct expanse scales. Further, whole genome expression profiling revealed marked shifts in the molecular topography with dynamic loading. These results demonstrate, for the first time, that dynamic compressive loading initiated after a sufficient period of chondro-induction and with sustained TGF-beta exposure enhances matrix distribution and the mechanical properties of MSC-seeded constructs.

  1. Hypoxia-Inducible Factor 1 Is an Inductor of Transcription Factor Activating Protein 2 Epsilon Expression during Chondrogenic Differentiation

    Directory of Open Access Journals (Sweden)

    Stephan Niebler

    2015-01-01

    Full Text Available The transcription factor AP-2ε (activating enhancer-binding protein epsilon is expressed in cartilage of humans and mice. However, knowledge about regulatory mechanisms influencing AP-2ε expression is limited. Using quantitative real time PCR, we detected a significant increase in AP-2ε mRNA expression comparing initial and late stages of chondrogenic differentiation processes in vitro and in vivo. Interestingly, in these samples the expression pattern of the prominent hypoxia marker gene angiopoietin-like 4 (Angptl4 strongly correlated with that of AP-2ε suggesting that hypoxia might represent an external regulator of AP-2ε expression in mammals. In order to show this, experiments directly targeting the activity of hypoxia-inducible factor-1 (HIF1, the complex mediating responses to oxygen deprivation, were performed. While the HIF1-activating compounds 2,2′-dipyridyl and desferrioxamine resulted in significantly enhanced mRNA concentration of AP-2ε, siRNA against HIF1α led to a significantly reduced expression rate of AP-2ε. Additionally, we detected a significant upregulation of the AP-2ε mRNA level after oxygen deprivation. In sum, these different experimental approaches revealed a novel role for the HIF1 complex in the regulation of the AP-2ε gene in cartilaginous cells and underlined the important role of hypoxia as an important external regulatory stimulus during chondrogenic differentiation modulating the expression of downstream transcription factors.

  2. IGF-1 Gene Transfer to Human Synovial MSCs Promotes Their Chondrogenic Differentiation Potential without Induction of the Hypertrophic Phenotype

    Directory of Open Access Journals (Sweden)

    Yasutoshi Ikeda

    2017-01-01

    Full Text Available Mesenchymal stem cell- (MSC- based therapy is a promising treatment for cartilage. However, repair tissue in general fails to regenerate an original hyaline-like tissue. In this study, we focused on increasing the expression levels for insulin-like growth factor-1 (IGF-1 to improve repair tissue quality. The IGF-1 gene was introduced into human synovial MSCs with a lentiviral vector and examined the levels of gene expression and morphological status of MSCs under chondrogenic differentiation condition using pellet cultures. The size of the pellets derived from IGF-1-MSCs were significantly larger than those of the control group. The abundance of glycosaminoglycan (GAG was also significantly higher in the IGF-1-MSC group. The histology of the IGF-1-induced pellets demonstrated similarities to hyaline cartilage without exhibiting features of a hypertrophic chondrocyte phenotype. Expression levels for the Col2A1 gene and protein were significantly higher in the IGF-1 pellets than in the control pellets, but expression levels for Col10, MMP-13, ALP, and Osterix were not higher. Thus, IGF-1 gene transfer to human synovial MSCs led to an improved chondrogenic differentiation capacity without the detectable induction of a hypertrophic or osteogenic phenotype.

  3. Neurotensin enhances estradiol induced DNA synthesis in immature rat uterus

    Energy Technology Data Exchange (ETDEWEB)

    Mistry, A.; Vijayan, E.

    1985-05-27

    Systemic administration of Neurotensin, a tridecapeptide, in immature rats treated with estradiol benzoate significantly enhances uterine DNA synthesis as reflected by the incorporation of /sup 3/H-thymidine. The peptide may have a direct action on the uterus. Substance P, a related peptide, had no effect on uterine DNA synthesis. 18 references, 4 tables.

  4. Estradiol levels in prepubertal boys and girls--analytical challenges

    DEFF Research Database (Denmark)

    Bay, Katrine; Andersson, Anna-Maria; Skakkebaek, Niels E

    2004-01-01

    Increasing evidence points at an important function of low concentrations of estradiol (E2) in prepubertal boys and girls. E2 serum levels in prepubertal children are, however, often immeasurable in conventional E2 assays. This strongly hampers further investigation of the physiological relevance...

  5. Effect of estradiol and oxytocin on ovine cervical relaxation

    African Journals Online (AJOL)

    Yomi

    2012-02-07

    Feb 7, 2012 ... The aim of this study was to examine the effect of estradiol (E2) and oxytocin ... Artificial insemination (AI) is a good way for the use of superior rams in reproduction but the conception rates in ... successful in sheep industry because it is costly, time .... during luteolysis and its abrogation in early pregnancy.

  6. 17 beta-estradiol affects osmoregulation in Fundulus heteroclitus

    NARCIS (Netherlands)

    Mancera, J.M.; Smolenaars, M.; Laiz-Carrion, R.; Rio, M. del; Wendelaar Bonga, S.E.; Flik, G.

    2004-01-01

    The effect of 17beta-estradiol (ED on osmoregulatory performance was examined in the euryhaline killifish, Fundulus heteroclitus. Fish were injected once with 1, 2 and 5 mug g(-1) E-2 and, 6 h after injection, transferred from I ppt seawater (SW) to full strength SW (40 ppt) or from SW to I ppt SW.

  7. Potential Biomedical Application of Enzymatically Treated Alginate/Chitosan Hydrosols in Sponges—Biocompatible Scaffolds Inducing Chondrogenic Differentiation of Human Adipose Derived Multipotent Stromal Cells

    Directory of Open Access Journals (Sweden)

    Anna Zimoch-Korzycka

    2016-08-01

    Full Text Available Current regenerative strategies used for cartilage repair rely on biomaterial functionality as a scaffold for cells that may have potential in chondrogenic differentiation. The purpose of the research was to investigate the biocompatibility of enzymatically treated alginate/chitosan hydrosol sponges and their suitability to support chondrogenic differentiation of human adipose derived multipotent stromal cells (hASCs. The alginate/chitosan and enzyme/alginate/chitosan sponges were formed from hydrosols with various proportions and were used as a biomaterial in this study. Sponges were tested for porosity and wettability. The porosity of each sponge was higher than 80%. An equal dose of alginate and chitosan in the composition of sponges improved their swelling ability. It was found that equal concentrations of alginate and chitosan in hydrosols sponges assure high biocompatibility properties that may be further improved by enzymatic treatment. Importantly, the high biocompatibility of these biomaterials turned out to be crucial in the context of hydrosols’ pro-chondrogenic function. After exposure to the chondrogenic conditions, the hASCs in N/A/C and L/A/C sponges formed well developed nodules and revealed increased expression of collagen type II, aggrecan and decreased expression of collagen type I. Moreover, in these cultures, the reactive oxygen species level was lowered while superoxide dismutase activity increased. Based on the obtained results, we conclude that N/A/C and L/A/C sponges may have prospective application as hASCs carriers for cartilage repair.

  8. The effects of the WNT-signaling modulators BIO and PKF118-310 on the chondrogenic differentiation of human mesenchymal stem cells

    NARCIS (Netherlands)

    Huang, Xiaobin; Zhong, Leilei; Hendriks, Jan; Post, Janine N.; Karperien, Marcel

    2018-01-01

    Mesenchymal stem cells (MSCs) are multipotent cells, mainly from bone marrow, and an ideal source of cells in bone and cartilage tissue engineering. A study of the chondrogenic differentiation of MSCs is of particular interest for MSCs-based cartilage regeneration. In this study, we aimed to

  9. Molecular Validation of Chondrogenic Differentiation and Hypoxia Responsiveness of Platelet-Lysate Expanded Adipose Tissue–Derived Human Mesenchymal Stromal Cells

    NARCIS (Netherlands)

    Galeano-Garces, Catalina; Camilleri, Emily T.; Riester, Scott M.; Dudakovic, Amel; Larson, Dirk R.; Qu, Wenchun; Smith, Jay; Dietz, Allan B.; Im, Hee-Jeong; Krych, Aaron J.; Larson, A. Noelle; Karperien, Marcel; van Wijnen, Andre J.

    2017-01-01

    Objective: To determine the optimal environmental conditions for chondrogenic differentiation of human adipose tissue–derived mesenchymal stromal/stem cells (AMSCs). In this investigation we specifically investigate the role of oxygen tension and 3-dimensional (3D) culture systems. Design: Both

  10. In-vivo generation of bone via endochondral ossification by in-vitro chondrogenic priming of adult human and rat mesenchymal stem cells

    LENUS (Irish Health Repository)

    Farrell, Eric

    2011-01-31

    Abstract Background Bone grafts are required to repair large bone defects after tumour resection or large trauma. The availability of patients\\' own bone tissue that can be used for these procedures is limited. Thus far bone tissue engineering has not lead to an implant which could be used as alternative in bone replacement surgery. This is mainly due to problems of vascularisation of the implanted tissues leading to core necrosis and implant failure. Recently it was discovered that embryonic stem cells can form bone via the endochondral pathway, thereby turning in-vitro created cartilage into bone in-vivo. In this study we investigated the potential of human adult mesenchymal stem cells to form bone via the endochondral pathway. Methods MSCs were cultured for 28 days in chondrogenic, osteogenic or control medium prior to implantation. To further optimise this process we induced mineralisation in the chondrogenic constructs before implantation by changing to osteogenic medium during the last 7 days of culture. Results After 8 weeks of subcutaneous implantation in mice, bone and bone marrow formation was observed in 8 of 9 constructs cultured in chondrogenic medium. No bone was observed in any samples cultured in osteogenic medium. Switch to osteogenic medium for 7 days prevented formation of bone in-vivo. Addition of β-glycerophosphate to chondrogenic medium during the last 7 days in culture induced mineralisation of the matrix and still enabled formation of bone and marrow in both human and rat MSC cultures. To determine whether bone was formed by the host or by the implanted tissue we used an immunocompetent transgenic rat model. Thereby we found that osteoblasts in the bone were almost entirely of host origin but the osteocytes are of both host and donor origin. Conclusions The preliminary data presented in this manuscript demonstrates that chondrogenic priming of MSCs leads to bone formation in vivo using both human and rat cells. Furthermore, addition of

  11. Metabolism of androstenone, 17β-estradiol and dihydrotestosterone in primary cultured pig hepatocytes and the role of 3β-hydroxysteroid dehydrogenase in this process.

    Directory of Open Access Journals (Sweden)

    Gang Chen

    Full Text Available Steroids metabolism plays an important role in mammals and contributes to quality of pig meat. The main objective of this study was to identify metabolites of androstenone, 17β-estradiol and dihydrotestosterone using primary cultured pig hepatocytes as a model system. The role of 3β-hydroxysteroid dehydrogenase (3βHSD in regulation of steroid metabolism was also validated using trilostane, a specific 3βHSD inhibitor. Steroid glucuronide conjugated metabolites were detected by liquid chromatography time of flight mass spectrometry (LC-TOF-MS. 3βHSD enzyme was essential for metabolism of androstenone to 5α-androst-16-en-3β-ol, which then formed androstenone glucuronide conjugate. Metabolism of 17β-estradiol was accompanied by formation of glucuronide-conjugated estrone and glucuronide-conjugated estradiol. The ratio of the two metabolites was around 5:1. 3βHSD enzyme was not involved in 17β-estradiol metabolism. 5α-Dihydrotestosterone-17β-glucuronide was identified as a dihydrotestosterone metabolite, and this metabolism was related to 3βHSD enzyme activity as demonstrated by inhibition study.

  12. Implication of C-type natriuretic peptide-3 signaling in glycosaminoglycan synthesis and chondrocyte hypertrophy during TGF-β1 induced chondrogenic differentiation of chicken bone marrow-derived mesenchymal stem cells.

    Science.gov (United States)

    Kocamaz, Erdogan; Gok, Duygu; Cetinkaya, Ayse; Tufan, A Cevik

    2012-10-01

    This study investigated the involvement of CNP-3, chick homologue for human C-type natriuretic peptide (CNP), in TGF-β1 induced chondrogenic differentiation of chicken bone marrow-derived mesenchymal stem cells (MSCs). Chondrogenic differentiation of MSCs in pellet cultures was induced by TGF-β1. Chondrogenic differentiation and glycosaminoglycan synthesis were analyzed on the basis of basic histology, collagen type II expression, and Alcian blue staining. Antibodies against CNP and NPR-B were used to block their function during these processes. Results revealed that expression of CNP-3 and NPR-B in MSCs were regulated by TGF-β1 in monolayer cultures at mRNA level. In pellet cultures of MSCs, TGF-β1 successfully induced chondrogenic differentiation and glycosaminoglycan synthesis. Addition of CNP into the TGF-β1 supplemented chondrogenic differentiation medium further induced the glycosaminoglycan synthesis and hypertrophy of differentiated chondrocytes in these pellets. Pellets induced with TGF-β1 and treated with antibodies against CNP and NPR-B, did show collagen type II expression, however, Alcian blue staining showing glycosaminoglycan synthesis was significantly suppressed. In conclusion, CNP-3/NPR-B signaling may strongly be involved in synthesis of glycosaminoglycans of the chondrogenic matrix and hypertrophy of differentiated chondrocytes during TGF-β1 induced chondrogenic differentiation of MSCs.

  13. Application of different 125I tracers in radioimmunoassays of estradiol-17β

    International Nuclear Information System (INIS)

    Bienert, R.; Flentje, H.; Herzmann, H.; Akademie der Wissenschaften der DDR, Leipzig. Zentralinstitut fuer Isotopen- und Strahlenforschung)

    1984-01-01

    Some different 125 I-labelled estradiol tracers were produced by direct radioiodizing of estradiol and also of the histamine and tyramine conjugates of estradiol-3-carboxymethylether (E 2 -3-CM) by means of the chloramine-T method. The linkage properties of these tracers were investigated in relation to the 3 H-labelled estradiol opposite to the antisera, which were produced against the cow serum albumin (RSA) conjugates of E 2 -3-CM and estradiol-6-carboxymethyloxime (E 2 -6-CMO). As suitable system for the radioimmunological estradiol determination could be revealed 4- 125 I-iodine estradiol in connection with one antiserum in each case of the radioligand antiserum combinations against E 2 -3-CM-RSA- and E 2 -6-CMO-RSA-conjugate. The double antibody method is used for separation in optimized RIA systems. The first and the second antibody reaction take place simultaneously. (author)

  14. The Infrapatellar Fat Pad as a Source of Perivascular Stem Cells with Increased Chondrogenic Potential for Regenerative Medicine.

    Science.gov (United States)

    Hindle, Paul; Khan, Nusrat; Biant, Leela; Péault, Bruno

    2017-01-01

    Perivascular stem cells (PSCs) are the natural ancestors of mesenchymal stem cells (MSCs) and are the stem cells responsible for homeostasis and repair in vivo. Prospectively identified and isolated PSCs have demonstrated increased plasticity and osteogenic potential. Cells from the infrapatellar fat pad (IFP) have demonstrated increased chondrogenic potential compared with those from subcutaneous fat. This research assessed the chondrogenic potential of IFP PSCs compared with MSCs from the IFP and bone marrow. Immunohistochemistry demonstrated the location of perivascular markers (CD146, CD34, neural/glial antigen 2 [NG2], platelet-derived growth factor receptor-β [PDGFRβ], and α-smooth muscle actin [α-SMA]) in relation to endothelial markers (CD31, CD144, von Willebrand factor [vWF]). Pericytes and adventitial cells were isolated from the stromal vascular fraction (3.8% and 21.2%, respectively) using flow cytometry with a viability of 88%. The mean numbers of pericytes and adventitial cells isolated were 4.6 ± 2.2 × 10 4 and 16.2 ± 3.2 × 10 4 , respectively, equating to 7.9 ± 4.4 × 10 3 and 20.8 ± 4.3 × 10 3 cells per gram of harvested tissue. Fluorescence-activated cell sorting demonstrated that cultured PSCs were CD44+CD90+CD105+; polymerase chain reaction and immunocytochemistry demonstrated that pericytes retained their CD146+ phenotype and expressed the pericyte markers PDGFRβ and NG2. Differentiation was confirmed using histochemical stains and genetic expression. Using a pellet model, the IFP PSCs and the MSCs generated significantly more extracellular matrix than bone marrow MSCs (p < .001 and p = .011, respectively). The IFP PSCs generated significantly more extracellular matrix than IFP MSCs (p = .002). Micromass culture demonstrated that differentiated PSCs were upregulated compared with MSCs for COL2A1, ACAN, and SOX9 expression by factors of 4.8 ± 1.3, 4.3 ± 0.9, and 7.0 ± 1.7, respectively. The IFP was a significantly better source

  15. The effects of dynamic and three-dimensional environments on chondrogenic differentiation of bone marrow stromal cells

    Energy Technology Data Exchange (ETDEWEB)

    Jung, Youngmee; Kim, Sang-Heon; Kim, Soo Hyun [Biomaterials Research Center, Korea Institute of Science and Technology, PO Box 131, Cheonryang, Seoul, 130-650 (Korea, Republic of); Kim, Young Ha, E-mail: soohkim@kist.re.k [Department of Materials Science and Engineering, Gwangju Institute of Science and Technology, 261 Cheomdan-gwagiro, Buk-gu, Gwangju 500-712 (Korea, Republic of)

    2009-10-15

    Articular cartilage is subjected to complex loading, which plays a major role in its growth, development and maintenance. Previously, we found that mechanical stimuli enhanced the development and function of engineered cartilage tissues in elastic mechano-active poly(lactide-co-caprolactone) (PLCL) scaffolds. In addition, it is well known that the three-dimensional spatial organization of cells and extracellular matrices in hydrogels is crucial to chondrogenesis. This study was conducted to enhance the chondrogenic differentiation of bone marrow stromal cells (BMSCs) in the hybrid scaffolds of fibrin gels and PLCL scaffolds in dynamic environments by compression. A highly elastic scaffold was fabricated from very elastic PLCL with 85% porosity and a 300-500{mu}m pore size using a gel-pressing method. A mixture of rabbit BMSCs and fibrin gels was then seeded onto the PLCL scaffolds and subjected to continuous compressive deformation of 5% strain at 0.1 Hz for 10 days in a chondrogenic medium containing 10 ng ml{sup -1} TGF-beta{sub 1}. The BMSCs-seeded scaffold constructs were then implanted subcutaneously into nude mice. As a control, the cell-PLCL scaffold constructs were cultured under dynamic conditions or the cell-PLCL/fibrin hybrid scaffold constructs and the cell-PLCL scaffold constructs were cultured under static conditions for 10 days in vitro. The results revealed that cells adhered onto the hybrid scaffolds of fibrin gels and PLCL scaffolds cultured under dynamic conditions. In addition, the accumulation of the extracellular matrix of cell-scaffold constructs, which was increased through mechanical stimulation, showed that chondrogenic differentiation was sustained and enhanced significantly in the stimulated hybrid scaffold constructs. Overall, the results of this study indicate that the proper periodic application of dynamic compression and the three-dimensional environments of the hybrid scaffolds composed of fibrin gels and elastic PLCL can encourage

  16. Estradiol and endocrine disrupting compounds adversely affect development of sea urchin embryos at environmentally relevant concentrations

    Energy Technology Data Exchange (ETDEWEB)

    Roepke, Troy A. [Bodega Marine Laboratory, University of California, Davis, POB 247, Bodega Bay, CA 94923 (United States); Snyder, Mark J. [Bodega Marine Laboratory, University of California, Davis, POB 247, Bodega Bay, CA 94923 (United States); Cherr, Gary N. [Bodega Marine Laboratory, University of California, Davis, POB 247, Bodega Bay, CA 94923 (United States) and Departments of Environmental Toxicology and Nutrition, One Shields Avenue, University of California, Davis, CA 95616 (United States)]. E-mail: gncherr@ucdavis.edu

    2005-01-26

    Environmental endocrine disrupting compounds (EDCs) are a wide variety of chemicals that typically exert effects, either directly or indirectly, through receptor-mediated processes, thus mimicking endogenous hormones and/or inhibiting normal hormone activities and metabolism. Little is known about the effects of EDCs on echinoderm physiology, reproduction and development. We exposed developing sea urchin embryos (Strongylocentrotus purpuratus and Lytechinus anamesus) to two known EDCs (4-octylphenol (OCT), bisphenol A (BisA)) and to natural and synthetic reproductive hormones (17{beta}-estradiol (E{sub 2}), estrone (E{sub 1}), estriol (E{sub 3}), progesterone (P{sub 4}) and 17{alpha}-ethynylestradiol (EE{sub 2})). In addition, we studied two non-estrogenic EDCs, tributyltin (TBT) and o,p-DDD. Successful development to the pluteus larval stage (96 h post-fertilization) was used to define EDC concentration-response relationships. The order of compound potency based on EC{sub 50} values for a reduction in normal development was as follows: TBT {sub L.anamesus} > OCT > TBT {sub S.{sub p}}{sub urpuratus} >> E{sub 2} > EE{sub 2} > DDD >> BisA > P{sub 4} > E{sub 1} >> E{sub 3}. The effect of TBT was pronounced even at concentrations substantially lower than those commonly reported in heavily contaminated areas, but the response was significantly different in the two model species. Sea urchin embryos were generally more sensitive to estrogenic EDCs and TBT than most other invertebrate larvae. Stage-specific exposure experiments were conducted to determine the most sensitive developmental periods using blastula, gastrula and post-gastrula (pluteus) stages. The stage most sensitive to E{sub 2}, OCT and TBT was the blastula stage with less overall sensitivity in the gastrula stage, regardless of concentration. Selective estrogen receptor modulators (SERMs) were added to the experiments individually and in combination with estrogenic EDCs to interfere with potential receptor

  17. Midazolam inhibits chondrogenesis via peripheral benzodiazepine receptor in human mesenchymal stem cells.

    Science.gov (United States)

    Chen, Yung-Ching; Wu, King-Chuen; Huang, Bu-Miin; So, Edmund Cheung; Wang, Yang-Kao

    2018-05-01

    Midazolam, a benzodiazepine derivative, is widely used for sedation and surgery. However, previous studies have demonstrated that Midazolam is associated with increased risks of congenital malformations, such as dwarfism, when used during early pregnancy. Recent studies have also demonstrated that Midazolam suppresses osteogenesis of mesenchymal stem cells (MSCs). Given that hypertrophic chondrocytes can differentiate into osteoblast and osteocytes and contribute to endochondral bone formation, the effect of Midazolam on chondrogenesis remains unclear. In this study, we applied a human MSC line, the KP cell, to serve as an in vitro model to study the effect of Midazolam on chondrogenesis. We first successfully established an in vitro chondrogenic model in a micromass culture or a 2D high-density culture performed with TGF-β-driven chondrogenic induction medium. Treatment of the Midazolam dose-dependently inhibited chondrogenesis, examined using Alcian blue-stained glycosaminoglycans and the expression of chondrogenic markers, such as SOX9 and type II collagen. Inhibition of Midazolam by peripheral benzodiazepine receptor (PBR) antagonist PK11195 or small interfering RNA rescued the inhibitory effects of Midazolam on chondrogenesis. In addition, Midazolam suppressed transforming growth factor-β-induced Smad3 phosphorylation, and this inhibitory effect could be rescued using PBR antagonist PK11195. This study provides a possible explanation for Midazolam-induced congenital malformations of the musculoskeletal system through PBR. © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  18. The effect of magnetic stimulation on the osteogenic and chondrogenic differentiation of human stem cells derived from the adipose tissue (hASCs)

    Energy Technology Data Exchange (ETDEWEB)

    Lima, João; Gonçalves, Ana I.; Rodrigues, Márcia T.; Reis, Rui L. [3Bs Research Group–Biomaterials, Biodegradables and Biomimetics, University of Minho, Guimarães (Portugal); ICVS/3Bs–PT Government Associate Laboratory, Braga/Guimarães (Portugal); Gomes, Manuela E., E-mail: megomes@dep.uminho.pt [3Bs Research Group–Biomaterials, Biodegradables and Biomimetics, University of Minho, Guimarães (Portugal); ICVS/3Bs–PT Government Associate Laboratory, Braga/Guimarães (Portugal)

    2015-11-01

    The use of magnetic nanoparticles (MNPs) towards the musculoskeletal tissues has been the focus of many studies, regarding MNPs ability to promote and direct cellular stimulation and orient tissue responses. This is thought to be mainly achieved by mechano-responsive pathways, which can induce changes in cell behavior, including the processes of proliferation and differentiation, in response to external mechanical stimuli. Thus, the application of MNP-based strategies in tissue engineering may hold potential to propose novel solutions for cell therapy on bone and cartilage strategies to accomplish tissue regeneration. The present work aims at studying the influence of MNPs on the osteogenic and chondrogenic differentiation of human adipose derived stem cells (hASCs). MNPs were incorporated in hASCs and cultured in medium supplemented for osteogenic and chondrogenic differentiation. Cultures were maintained up to 28 days with/without an external magnetic stimulus provided by a magnetic bioreactor, to determine if the MNPs alone could affect the osteogenic or chondrogenic phenotype of the hASCs. Results indicate that the incorporation of MNPs does not negatively affect the viability nor the proliferation of hASCs. Furthermore, Alizarin Red staining evidences an enhancement in extracellular (ECM) mineralization under the influence of an external magnetic field. Although not as evident as for osteogenic differentiation, Toluidine blue and Safranin-O stainings also suggest the presence of a cartilage-like ECM with glycosaminoglycans and proteoglycans under the magnetic stimulus provided. Thus, MNPs incorporated in hASCs under the influence of an external magnetic field have the potential to induce differentiation towards the osteogenic and chondrogenic lineages. - Highlights: • Cellular viability was not negatively influenced by the nanoparticles. • Chondrogenic medium influences more the synthesis of cartilage-like ECM than MNPs. • Synergetic effect among

  19. TGF-β1 is Involved in Vitamin D-Induced Chondrogenic Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells by Regulating the ERK/JNK Pathway

    Directory of Open Access Journals (Sweden)

    Xiaorui Jiang

    2017-08-01

    Full Text Available Background/Aims: Osteoarthritis (OA is characterized by degradation of cartilage, sole cell type of which is chondrocytes. Bone marrow-derived mesenchymal stem cells (BMSCs possess multipotency and can be directionally differentiated into chondrocytes under stimulation. This study was aimed to explore the possible roles of vitamin D and transforming growth factor-β1 (TGF-β1 in the chondrogenic differentiation of BMSCs. Methods: BMSCs were isolated from femurs and tibias of rats and characterized by flow cytometry. After stimulation with vitamin D, BMSC proliferation and migration were measured by Cell Counting Kit-8 (CCK-8 and Transwell assays, respectively. Chondrogenic differentiation was estimated through expression levels of specific markers by qRT-PCR and Western blot analysis. After stable transfection, the effects of aberrantly expressed TGF-β1 on vitamin D-induced alterations, including BMSC viability, migration and chondrogenic differentiation, were all evaluated utilizing CCK-8 assay, Transwell assay, qRT-PCR and Western blot analysis. Finally, the phosphorylation levels of key kinases in the extracellular signal-regulated kinase (ERK and c-Jun N-terminal kinase (JNK pathways were determined by Western blot analysis. Results: Vitamin D remarkably promoted BMSC viability, migration and chondrogenic differentiation. These alterations of BMSCs induced by vitamin D were reinforced by TGF-β1 overexpression while were reversed by TGF-β1 silencing. Additionally, the phosphorylation levels of ERK, JNK and c-Jun were enhanced by TGF-β1 overexpression but were reduced by TGF-β1 knockdown. Conclusion: Vitamin D promoted BMSC proliferation, migration and chondrogenic differentiation. TGF-β1 might be implicated in the vitamin D-induced alterations of BMSCs through regulating ERK/JNK pathway.

  20. The effect of magnetic stimulation on the osteogenic and chondrogenic differentiation of human stem cells derived from the adipose tissue (hASCs)

    International Nuclear Information System (INIS)

    Lima, João; Gonçalves, Ana I.; Rodrigues, Márcia T.; Reis, Rui L.; Gomes, Manuela E.

    2015-01-01

    The use of magnetic nanoparticles (MNPs) towards the musculoskeletal tissues has been the focus of many studies, regarding MNPs ability to promote and direct cellular stimulation and orient tissue responses. This is thought to be mainly achieved by mechano-responsive pathways, which can induce changes in cell behavior, including the processes of proliferation and differentiation, in response to external mechanical stimuli. Thus, the application of MNP-based strategies in tissue engineering may hold potential to propose novel solutions for cell therapy on bone and cartilage strategies to accomplish tissue regeneration. The present work aims at studying the influence of MNPs on the osteogenic and chondrogenic differentiation of human adipose derived stem cells (hASCs). MNPs were incorporated in hASCs and cultured in medium supplemented for osteogenic and chondrogenic differentiation. Cultures were maintained up to 28 days with/without an external magnetic stimulus provided by a magnetic bioreactor, to determine if the MNPs alone could affect the osteogenic or chondrogenic phenotype of the hASCs. Results indicate that the incorporation of MNPs does not negatively affect the viability nor the proliferation of hASCs. Furthermore, Alizarin Red staining evidences an enhancement in extracellular (ECM) mineralization under the influence of an external magnetic field. Although not as evident as for osteogenic differentiation, Toluidine blue and Safranin-O stainings also suggest the presence of a cartilage-like ECM with glycosaminoglycans and proteoglycans under the magnetic stimulus provided. Thus, MNPs incorporated in hASCs under the influence of an external magnetic field have the potential to induce differentiation towards the osteogenic and chondrogenic lineages. - Highlights: • Cellular viability was not negatively influenced by the nanoparticles. • Chondrogenic medium influences more the synthesis of cartilage-like ECM than MNPs. • Synergetic effect among

  1. Human platelet lysate successfully promotes proliferation and subsequent chondrogenic differentiation of adipose-derived stem cells: a comparison with articular chondrocytes.

    Science.gov (United States)

    Hildner, F; Eder, M J; Hofer, K; Aberl, J; Redl, H; van Griensven, M; Gabriel, C; Peterbauer-Scherb, A

    2015-07-01

    Fetal calf serum (FCS) bears a potential risk for carrying diseases and eliciting immune reactions. Nevertheless, it still represents the gold standard as medium supplement in cell culture. In the present study, human platelet lysate (PL) was tested as an alternative to FCS for the expansion and subsequent chondrogenic differentiation of human adipose-derived stem cells (ASCs). ASCs were expanded with 10% FCS (group F) or 5% PL (group P). Subsequently, three-dimensional (3D) micromass pellets were created and cultured for 5 weeks in chondrogenic differentiation medium. Additionally, the de- and redifferentiation potential of human articular chondrocytes (HACs) was evaluated and compared to ASCs. Both HACs and ASCs cultured with PL showed strongly enhanced proliferation rates. Redifferentiation of HACs was possible for cells expanded up to 3.3 population doublings (PD). At this stage, PL-expanded HACs demonstrated better redifferentiation potential than FCS-expanded cells. ASCs could also be differentiated following extended passaging. Glycosaminoglycan (GAG) quantification and qRT-PCR of 10 cartilage related markers demonstrated a tendency for increased chondrogenic differentiation of PL-expanded ASCs compared to cells expanded with FCS. Histologically, collagen type II but also collagen type X was mainly present in group P. The present study demonstrates that PL strongly induces proliferation of ASCs, while the chondrogenic differentiation potential is retained. HACs also showed enhanced proliferation and even better redifferentiation when previously expanded with PL. This suggests that PL is superior to FCS as a supplement for the expansion of ASCs and HACs, particularly with regard to chondrogenic (re)differentiation. Copyright © 2013 John Wiley & Sons, Ltd.

  2. Baicalein has protective effects on the 17β-estradiol-induced transformation of breast epithelial cells.

    Science.gov (United States)

    Chen, Yan; Wang, Jing; Hong, Duan-Yang; Chen, Lin; Zhang, Yan-Yan; Xu, Yi-Ni; Pan, Di; Fu, Ling-Yun; Tao, Ling; Luo, Hong; Shen, Xiang-Chun

    2017-02-07

    Epidemiologic and systematic studies have indicated that flavonoid consumption is associated with a lower incidence of breast cancer. Baicalein is the primary flavonoid derived from the roots of Scutellaria baicalensis Georgi. In the current study, the long-term exposure of breast epithelial cells to 17β-estradiol (E2) was used to investigate the chemopreventive potential of baicalein on neoplastic transformation. The results demonstrated that baicalein significantly inhibited E2-induced cell growth, motility, and invasiveness, and suppressed E2-induced misshapen acini formation in 3D cultures. Furthermore, it inhibited the ability of E2-induced cells to form clones in agarose and tumors in NOD/SCID immunodeficient mice. Docking studies using Sybyl-X 1.2 software showed that baicalein could bind to both estrogen receptor-α (ERa) and G-protein coupled estrogen receptor 30 (GPR30), which are two critical E2-mediated pathways. Baicalein prevented the E2-induced ERa-mediated activation of nuclear transcriptional signaling by interfering with the trafficking of ERa into the nucleus and subsequent binding to estrogen response elements, thereby decreasing the mRNA levels of ERa target genes. It also inhibited E2-induced GPR30-mediated signal transduction, as well as the transcription of GPR30-regulated genes. Therefore, these results suggest that baicalein is a potential drug for reducing the risk of estrogen-dependent breast cancer.

  3. Collagen-Coated Polytetrafluoroethane Membrane Inserts Enhances Chondrogenic Differentiation of Human Cord Blood Multi-Lineage Progenitor Cells

    DEFF Research Database (Denmark)

    Munir, Samir; Søballe, Kjeld; Ulrich-Vinther, Michael

    culturing resulted in a multicellular layer tissue with formation of more cartilaginous tissue compared to micromass or CPP culture. In the membrane system MLPCs produced pellucid discs, 12 mm in diameter by 1 mm in thickness from 2x10^6 cells. The discs had hyaline-like cartilage extracellular matrix......Background: Articular chondrocytes and bone marrow-derived multipotent mesenchymal stromal cells (MSCs) are the favoured cells for cartilage tissue engineering. Umbilical cord blood has proven an alternative source of MSCs and moreover they may be more potent chondroprogenitor cells than bonemarrow...... with micromass or CPP cultures. Conclusions: In conclusion, we demonstrate that MLPCs possess’ chondrogenic potency, which increased when cultured scaffold-free on membrane inserts resulting in multicellular-layered hyaline-like cartilage tissue. Evaluating the effect of culturing pre-differentiated MLPCs on CPP...

  4. Poly(γ-Glutamic Acid) as an Exogenous Promoter of Chondrogenic Differentiation of Human Mesenchymal Stem/Stromal Cells.

    Science.gov (United States)

    Antunes, Joana C; Tsaryk, Roman; Gonçalves, Raquel M; Pereira, Catarina Leite; Landes, Constantin; Brochhausen, Christoph; Ghanaati, Shahram; Barbosa, Mário A; Kirkpatrick, C James

    2015-06-01

    Cartilage damage and/or aging effects can cause constant pain, which limits the patient's quality of life. Although different strategies have been proposed to enhance the limited regenerative capacity of cartilage tissue, the full production of native and functional cartilaginous extracellular matrix (ECM) has not yet been achieved. Poly(γ-glutamic acid) (γ-PGA), a naturally occurring polyamino acid, biodegradable into glutamate residues, has been explored for tissue regeneration. In this work, γ-PGA's ability to support the production of cartilaginous ECM by human bone marrow mesenchymal stem/stromal cells (MSCs) and nasal chondrocytes (NCs) was investigated. MSC and NC pellets were cultured in basal medium (BM), chondrogenic medium (CM), and CM-γ-PGA-supplemented medium (CM+γ-PGA) over a period of 21 days. Pellet size/shape was monitored with time. At 14 and 21 days of culture, the presence of sulfated glycosaminoglycans (sGAGs), type II collagen (Col II), Sox-9, aggrecan, type XI collagen (Col XI), type X collagen (Col X), calcium deposits, and type I collagen (Col I) was analyzed. After excluding γ-PGA's cytotoxicity, earlier cell condensation, higher sGAG content, Col II, Sox-9 (day 14), aggrecan, and Col X (day 14) production was observed in γ-PGA-supplemented MSC cultures, with no signs of mineralization or Col I. These effects were not evident with NCs. However, Sox-9 (at day 14) and Col X (at days 14 and 21) were increased, decreased, or absent, respectively. Overall, γ-PGA improved chondrogenic differentiation of MSCs, increasing ECM production earlier in culture. It is proposed that γ-PGA incorporation in novel biomaterials has a beneficial impact on future approaches for cartilage regeneration.

  5. Fibroblast-Derived Extracellular Matrix Induces Chondrogenic Differentiation in Human Adipose-Derived Mesenchymal Stromal/Stem Cells in Vitro

    Directory of Open Access Journals (Sweden)

    Kevin Dzobo

    2016-08-01

    Full Text Available Mesenchymal stromal/stem cells (MSCs represent an area being intensively researched for tissue engineering and regenerative medicine applications. MSCs may provide the opportunity to treat diseases and injuries that currently have limited therapeutic options, as well as enhance present strategies for tissue repair. The cellular environment has a significant role in cellular development and differentiation through cell–matrix interactions. The aim of this study was to investigate the behavior of adipose-derived MSCs (ad-MSCs in the context of a cell-derived matrix so as to model the in vivo physiological microenvironment. The fibroblast-derived extracellular matrix (fd-ECM did not affect ad-MSC morphology, but reduced ad-MSC proliferation. Ad-MSCs cultured on fd-ECM displayed decreased expression of integrins α2 and β1 and subsequently lost their multipotency over time, as shown by the decrease in CD44, Octamer-binding transcription factor 4 (OCT4, SOX2, and NANOG gene expression. The fd-ECM induced chondrogenic differentiation in ad-MSCs compared to control ad-MSCs. Loss of function studies, through the use of siRNA and a mutant Notch1 construct, revealed that ECM-mediated ad-MSCs chondrogenesis requires Notch1 and β-catenin signaling. The fd-ECM also showed anti-senescence effects on ad-MSCs. The fd-ECM is a promising approach for inducing chondrogenesis in ad-MSCs and chondrogenic differentiated ad-MSCs could be used in stem cell therapy procedures.

  6. Membrane-Initiated Estradiol Signaling Regulating Sexual Receptivity

    Science.gov (United States)

    Micevych, Paul E.; Dewing, Phoebe

    2011-01-01

    Estradiol has profound actions on the structure and function of the nervous system. In addition to nuclear actions that directly modulate gene expression, the idea that estradiol can rapidly activate cell signaling by binding to membrane estrogen receptors (mERs) has emerged. Even the regulation of sexual receptivity, an action previously thought to be completely regulated by nuclear ERs, has been shown to have a membrane-initiated estradiol signaling (MIES) component. This highlighted the question of the nature of mERs. Several candidates have been proposed, ERα, ERβ, ER-X, GPR30 (G protein coupled estrogen receptor), and a receptor activated by a diphenylacrylamide compound, STX. Although each of these receptors has been shown to be active in specific assays, we present evidence for and against their participation in sexual receptivity by acting in the lordosis-regulating circuit. The initial MIES that activates the circuit is in the arcuate nucleus of the hypothalamus (ARH). Using both activation of μ-opioid receptors (MOR) in the medial preoptic nucleus and lordosis behavior, we document that both ERα and the STX-receptor participate in the required MIES. ERα and the STX-receptor activation of cell signaling are dependent on the transactivation of type 1 metabotropic glutamate receptors (mGluR1a) that augment progesterone synthesis in astrocytes and protein kinase C (PKC) in ARH neurons. While estradiol-induced sexual receptivity does not depend on neuroprogesterone, proceptive behaviors do. Moreover, the ERα and the STX-receptor activation of medial preoptic MORs and augmentation of lordosis were sensitive to mGluR1a blockade. These observations suggest a common mechanism through which mERs are coupled to intracellular signaling cascades, not just in regulating reproduction, but in actions throughout the neuraxis including the cortex, hippocampus, striatum, and dorsal root ganglias. PMID:22649369

  7. Membrane–initiated estradiol signaling regulating sexual receptivity

    Directory of Open Access Journals (Sweden)

    Paul E Micevych

    2011-09-01

    Full Text Available Estradiol has profound actions on the structure and function of the nervous system. In addition to nuclear actions that directly modulate gene expression, the idea that estradiol can rapidly activate cell signaling by binding to membrane estrogen receptors (mERs has emerged. Even the regulation of sexual receptivity, an action previously thought to be completely regulated by nuclear ERs, has been shown to have a membrane-initiated estradiol signaling (MIES component. This highlighted the question of the nature of mERs. Several candidates have been proposed, ERα, ERβ, ER-X, GPR30 (G protein coupled estrogen receptor; GPER, and a receptor activated by a diphenylacrylamide compound, STX. Although each of these receptors has been shown to be active in specific assays, we present evidence for and against their participation in sexual receptivity by acting in the lordosis-regulating circuit. The initial MIES that activates the circuit is in the arcuate nucleus of the hypothalamus (ARH. Using both activation of μ-opioid receptors (MOR in the medial preoptic nucleus and lordosis behavior, we document that both ERα and the STX receptor participate in the required MIES. ERα and the STX receptor activation of cell signaling are dependent on the transactivation of type 1 metabotropic glutamate receptors (mGluR1a that augment progesterone synthesis in astrocytes and protein kinase C (PKC in ARH neurons. While estradiol-induced sexual receptivity does not depend on neuroprogesterone, proceptive behaviors do. Moreover, the ERα and the STX receptor activation of medial preoptic MORs and augmentation of lordosis were sensitive to mGluR1a blockade. These observations suggest a common mechanism through which mERs are coupled to intracellular signaling cascades, not just in regulating reproduction, but in actions throughout the neuraxis including the cortex, hippocampus, striatum and DRGs.

  8. L-Type Calcium Channels Modulation by Estradiol.

    Science.gov (United States)

    Vega-Vela, Nelson E; Osorio, Daniel; Avila-Rodriguez, Marco; Gonzalez, Janneth; García-Segura, Luis Miguel; Echeverria, Valentina; Barreto, George E

    2017-09-01

    Voltage-gated calcium channels are key regulators of brain function, and their dysfunction has been associated with multiple conditions and neurodegenerative diseases because they couple membrane depolarization to the influx of calcium-and other processes such as gene expression-in excitable cells. L-type calcium channels, one of the three major classes and probably the best characterized of the voltage-gated calcium channels, act as an essential calcium binding proteins with a significant biological relevance. It is well known that estradiol can activate rapidly brain signaling pathways and modulatory/regulatory proteins through non-genomic (or non-transcriptional) mechanisms, which lead to an increase of intracellular calcium that activate multiple kinases and signaling cascades, in the same way as L-type calcium channels responses. In this context, estrogens-L-type calcium channels signaling raises intracellular calcium levels and activates the same signaling cascades in the brain probably through estrogen receptor-independent modulatory mechanisms. In this review, we discuss the available literature on this area, which seems to suggest that estradiol exerts dual effects/modulation on these channels in a concentration-dependent manner (as a potentiator of these channels in pM concentrations and as an inhibitor in nM concentrations). Indeed, estradiol may orchestrate multiple neurotrophic responses, which open a new avenue for the development of novel estrogen-based therapies to alleviate different neuropathologies. We also highlight that it is essential to determine through computational and/or experimental approaches the interaction between estradiol and L-type calcium channels to assist these developments, which is an interesting area of research that deserves a closer look in future biomedical research.

  9. Nomegestrol acetate-17b-estradiol for oral contraception

    Directory of Open Access Journals (Sweden)

    Burke A

    2013-06-01

    Full Text Available Anne Burke Johns Hopkins University School of Medicine, Baltimore, MD, USAAbstract: Oral contraceptives remain a popular method of contraception over 50 years after their introduction. While safe and effective for many women, the failure rate of oral contraception is about 8%. Concerns about the risk of venous thromboembolism continue to drive the search for the safest oral contraceptive formulations. The oral contraceptive NOMAC-E2 contains nomegestrol acetate (NOMAC 2.5 mg + 17b-estradiol (E2 1.5 mg. The approved dosing regimen is 24 days of active hormone, followed by a 4-day hormone-free interval. NOMAC is a progestin derived from testosterone, which has high bioavailability, rapid absorption, and a long half-life. Estradiol, though it has a lower bioavailability, has been successfully combined with NOMAC in a monophasic oral contraceptive. Two recently published randomized controlled trials demonstrate that NOMAC-E2 is an effective contraceptive, with a Pearl Index less than one pregnancy per 100 woman-years. The bleeding pattern on NOMAC-E2 is characterized by fewer bleeding/spotting days, shorter withdrawal bleeds, and a higher incidence of amenorrhea than the comparator oral contraceptive containing drospirenone and ethinyl estradiol. The adverse event profile appears to be acceptable. Few severe adverse events were reported in the randomized controlled trials. The most common adverse events were irregular bleeding, acne, and weight gain. Preliminary studies suggest that NOMAC-E2 does not seem to have negative effects on hemostatic and metabolic parameters. While no one oral contraceptive formulation is likely to be the optimum choice for all women, NOMAC-E2 is a formulation with effectiveness comparable with that of other oral contraceptives, and a reassuring safety profile.Keywords: oral contraception, nomegestrol acetate, estradiol

  10. Metabolic clearance and blood production rates of estradiol in hyperthyroidism.

    Science.gov (United States)

    Ridgway, E C; Longcope, C; Maloof, F

    1975-09-01

    The metabolic clearance rate of 17beta-estradiol (MCR2), the plasma levels of 17beta-estradiol (E2)1, sex-steroid binding globulin (SSBG), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured in 10 hyperthyroid subjects (7 men and 3 women). The blood production rate of 17beta-estradiol (PB2) was calculated for all subjects. Nine of the 10 hyperthyroid subjects had a decreased MCR2 which returned towards normal in 5 of the 6 subjects restudied following therapy. In all 10 subjects the levels of SSBG were increased when they were hyperthyroid and returned toward normal with therapy. It is concluded that the decrease in MCR2 is largely due to the increased binding of 17beta-estradiol to SSBG. In 7 of the 10 hyperthyroid the plasma E2 concentrations were normal whereas 3 had slightly elevated levels. In 8 of the 10 hyperthyroid the PB2 was within the normal range. Only 2 hyperthyroid subjects had slightly elevated PB2. In the 6 subjects who were restudied after therapy, there was no consistent change in PB2 which remained in the normal range in all cases. It is concluded that the MCR2 is decreased in most subjects with hyperthyroidism in association with an increase of SSBG. Despite this change in MCR2 there is no significant change in PB2. The increase in SSBG levels in hyperthyroidism appears to be a direct effect of the elevation of thyroid hormone activity and is not mediated through estrogen.

  11. Basal and dynamic relationships between implicit power motivation and estradiol in women.

    Science.gov (United States)

    Stanton, Steven J; Schultheiss, Oliver C

    2007-12-01

    This study investigated basal and reciprocal relationships between implicit power motivation (n Power), a preference for having impact and dominance over others, and both salivary estradiol and testosterone in women. 49 participants completed the Picture Story Exercise, a measure of n Power. During a laboratory contest, participants competed in pairs on a cognitive task and contest outcome (win vs. loss) was experimentally varied. Estradiol and testosterone levels were determined in saliva samples collected at baseline and several times post-contest, including 1 day post-contest. n Power was positively associated with basal estradiol concentrations. The positive correlation between n Power and basal estradiol was stronger in single women, women not taking oral contraceptives, or in women with low-CV estradiol samples than in the overall sample of women. Women's estradiol responses to a dominance contest were influenced by the interaction of n Power and contest outcome: estradiol increased in power-motivated winners but decreased in power-motivated losers. For power-motivated winners, elevated levels of estradiol were still present the day after the contest. Lastly, n Power and estradiol did not correlate with self-reported dominance and correlated negatively with self-reported aggression. Self-reported dominance and aggression did not predict estradiol changes as a function of contest outcome. Overall, n Power did not predict basal testosterone levels or testosterone changes as a function of dominance contest outcome.

  12. The modulatory effect of estradiol benzoate on superoxide dismutase activity in the developing rat brain

    Directory of Open Access Journals (Sweden)

    Pejic S.

    2003-01-01

    Full Text Available The sensitivity of copper,zinc (CuZn- and manganese (Mn-superoxide dismutase (SOD to exogenous estradiol benzoate (EB was investigated in Wistar rats during postnatal brain development. Enzyme activities were measured in samples prepared from brains of rats of both sexes and various ages between 0 and 75 days, treated sc with 0.5 µg EB/100 g body weight in 0.1 ml olive oil/100 g body weight, 48 and 24 h before sacrifice. In females, EB treatment stimulated MnSOD activity on days 0 (66.1%, 8 (72.7% and 15 (81.7%. In males, the stimulatory effect of EB on MnSOD activity on day 0 (113.6% disappeared on day 8 and on days 15 and 45 it became inhibitory (40.3 and 30.5%, respectively. EB had no effect on the other age groups. The stimulatory effect of EB on CuZnSOD activity in newborn females (51.8% changed to an inhibitory effect on day 8 (38.4% and disappeared by day 45 when inhibition was detected again (48.7%. In males, the inhibitory effect on this enzyme was observed on days 0 (45.0% and 15 (28.9%, and then disappeared until day 60 when a stimulatory effect was observed (38.4%. EB treatment had no effect on the other age groups. The sensitivity of MnSOD to estradiol differed significantly between sexes during the neonatal and prepubertal period, whereas it followed a similar pattern thereafter. The sensitivity of CuZnSOD to estradiol differed significantly between sexes during most of the study period. Regression analysis showed that the sensitivity of MnSOD to this estrogen tended to decrease similarly in both sexes, whereas the sensitivity of CuZnSOD showed a significantly different opposite tendency in female and male rats. These are the first reports indicating hormonal modulation of antioxidant enzyme activities related to the developmental process.

  13. Maternal fructose intake disturbs ovarian estradiol synthesis in rats.

    Science.gov (United States)

    Munetsuna, Eiji; Yamada, Hiroya; Yamazaki, Mirai; Ando, Yoshitaka; Mizuno, Genki; Ota, Takeru; Hattori, Yuji; Sadamoto, Nao; Suzuki, Koji; Ishikawa, Hiroaki; Hashimoto, Shuji; Ohashi, Koji

    2018-06-01

    Recent increases in fructose consumption have raised concerns regarding the potential adverse intergenerational effects, as maternal fructose intake may induce physiological dysfunction in offspring. However, no reports are available regarding the effect of excess maternal fructose on reproductive tissues such as the ovary. Notably, the maternal intrauterine environment has been demonstrated to affect ovarian development in the subsequent generation. Given the fructose is transferred to the fetus, excess fructose consumption may affect offspring ovarian development. As ovarian development and its function is maintained by 17β-estradiol, we therefore investigated whether excess maternal fructose intake influences offspring ovarian estradiol synthesis. Rats received a 20% fructose solution during gestation and lactation. After weaning, offspring ovaries were isolated. Offspring from fructose-fed dams showed reduced StAR and P450(17α) mRNA levels, along with decreased protein expression levels. Conversely, attenuated P450arom protein level was found in the absence of mRNA expression alteration. Consistent with these phenomena, decreased circulating levels of estradiol were observed. Furthermore, estrogen receptor α (ERα) protein levels were also down-regulated. In accordance, the mRNA for progesterone receptor, a transcriptional target of ERα, was decreased. These results suggest that maternal fructose might alter ovarian physiology in the subsequent generation. Copyright © 2018 Elsevier Inc. All rights reserved.

  14. Estradiol upregulates progesterone receptor and orphanin FQ colocalization in arcuate nucleus neurons and opioid receptor-like receptor-1 expression in proopiomelanocortin neurons that project to the medial preoptic nucleus in the female rat

    Science.gov (United States)

    Sanathara, Nayna M.; Moreas, Justine; Mahavongtrakul, Matthew; Sinchak, Kevin

    2014-01-01

    Background Ovarian steroids regulate sexual receptivity in the female rat by acting on neurons that converge on proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (ARH) that project to the medial preoptic nucleus (MPN). Estradiol rapidly activates these neurons to release β-endorphin that activates MPN μ-opioid receptors (MOP) to inhibit lordosis. Lordosis is facilitated by the subsequent action of progesterone that deactivates the estradiol-induced MPN MOP activation. Orphanin FQ (OFQ/N; aka nociceptin) infusions into the ARH, like progesterone, deactivate MPN MOP and facilitate lordosis in estradiol-primed rats. OFQ/N reduces the activity of ARH β-endorphin neurons through post- and presynaptic mechanisms via its cognate receptor, ORL-1. Methods We tested the hypotheses that progesterone receptors (PR) are expressed in ARH OFQ/N neurons by immunohistochemistry and ORL-1 is expressed in POMC neurons that project to the MPN by combining Fluoro-Gold injection into the MPN and double-label fluorescent in situ hybridization (FISH). We also hypothesized that estradiol increases coexpression of PR-OFQ/N and ORL-1-POMC in ARH neurons of ovariectomized rats. Results The number of PR and OFQ/N immunopositive ARH neurons was increased as was their colocalization by estradiol treatment. FISH for ORL-1 and POMC mRNA revealed a subpopulation of ARH neurons that was triple-labeled indicating these neurons project to the MPN and coexpress ORL-1 and POMC mRNA. Estradiol was shown to upregulate ORL-1 and POMC expression in MPN-projecting ARH neurons. Conclusion Estradiol upregulates the ARH OFQ/N-ORL-1 system projecting to the MPN that regulates lordosis. PMID:24821192

  15. Prostate enlargement in mice due to fetal exposure to low doses of estradiol or diethylstilbestrol and opposite effects at high doses

    Science.gov (United States)

    Saal, Frederick S. vom; Timms, Barry G.; Montano, Monica M.; Palanza, Paola; Thayer, Kristina A.; Nagel, Susan C.; Dhar, Minati D.; Ganjam, V. K.; Parmigiani, Stefano; Welshons, Wade V.

    1997-01-01

    On the basis of results of studies using high doses of estrogens, exposure to estrogen during fetal life is known to inhibit prostate development. However, it is recognized in endocrinology that low concentrations of a hormone can stimulate a tissue, while high concentrations can have the opposite effect. We report here that a 50% increase in free-serum estradiol in male mouse fetuses (released by a maternal Silastic estradiol implant) induced a 40% increase in the number of developing prostatic glands during fetal life; subsequently, in adulthood, the number of prostatic androgen receptors per cell was permanently increased by 2-fold, and the prostate was enlarged by 30% (due to hyperplasia) relative to untreated males. However, as the free serum estradiol concentration in male fetuses was increased from 2- to 8-fold, adult prostate weight decreased relative to males exposed to the 50% increase in estradiol. As a model for fetal exposure to man-made estrogens, pregnant mice were fed diethylstilbestrol (DES) from gestation days 11 to 17. Relative to controls, DES doses of 0.02, 0.2, and 2.0 ng per g of body weight per day increased adult prostate weight, whereas a 200-ng-per-g dose decreased adult prostate weight in male offspring. Our findings suggest that a small increase in estrogen may modulate the action of androgen in regulating prostate differentiation, resulting in a permanent increase in prostatic androgen receptors and prostate size. For both estradiol and DES, prostate weight first increased then decreased with dose, resulting in an inverted-U dose-response relationship. PMID:9050904

  16. Histaminergic responses by hypothalamic neurons that regulate lordosis and their modulation by estradiol.

    Science.gov (United States)

    Dupré, Christophe; Lovett-Barron, Matthew; Pfaff, Donald W; Kow, Lee-Ming

    2010-07-06

    How do fluctuations in the level of generalized arousal of the brain affect the performance of specific motivated behaviors, such as sexual behaviors that depend on sexual arousal? A great deal of previous work has provided us with two important starting points in answering this question: (i) that histamine (HA) serves generalized CNS arousal and (ii) that heightened electrical activity of neurons in the ventromedial nucleus of the hypothalamus (VMN) is necessary and sufficient for facilitating the primary female sex behavior in laboratory animals, lordosis behavior. Here we used patch clamp recording technology to analyze HA effects on VMN neuronal activity. The results show that HA acting through H1 receptors (H1R) depolarizes these neurons. Further, acute administration of estradiol, an estrogen necessary for lordosis behavior to occur, heightens this effect. Hyperpolarization, which tends to decrease excitability and enhance inhibition, was not affected by acute estradiol or mediated by H1R but was mediated by other HA receptor subtypes, H2 and H3. Sampling of mRNA from individual VMN neurons showed colocalization of expression of H1 receptor mRNA with estrogen receptor (ER)-alpha mRNA but also revealed ER colocalization with the other HA receptor subtypes and colocalization of different subtypes with each other. The latter finding provides the molecular basis for complex "push-pull" regulation of VMN neuronal excitability by HA. Thus, in the simplest causal route, HA, acting on VMN neurons through H1R provides a mechanism by which elevated states of generalized CNS arousal can foster a specific estrogen-dependent, aroused behavior, sexual behavior.

  17. 17β-estradiol-induced growth of triple-negative breast cancer cells is prevented by the reduction of GPER expression after treatment with gefitinib.

    Science.gov (United States)

    Girgert, Rainer; Emons, Günter; Gründker, Carsten

    2017-02-01

    Triple-negative breast cancers (TNBCs) are neither susceptible to endocrine therapy due to a lack of estrogen receptor α expression nor trastuzumab. TNBCs frequently overexpress epidermal growth factor receptor (EGFR) and membrane bound estrogen receptor, GPER. To a certain extent the growth of TNBCs is stimulated by 17β-estradiol via GPER. We analyzed whether inhibition of EGFR by gefitinib reduces the expression of GPER and subsequent signal transduction in TNBC cells. Dependence of proliferation on 17β-estradiol was determined using Alamar Blue assay. Expression of GPR30 and activation of c-src, EGFR and cAMP-responsive element binding (CREB) protein by 17β-estradiol was analyzed by western blotting. Expression of c-fos, cyclin D1 and aromatase was determined using RT-PCR. Gefitinib reduced GPER expression concentration‑ and time‑dependently. In HCC70 cells, GPER expression was reduced to 15±11% (p<0.05) after treatment with 200 nM gefitinib for four days, and in HCC1806 cells GPER expression was reduced to 39±5% (p<0.01) of the control. 17β-estradiol significantly increased the percentage of HCC1806 cells within 7 days to 145±29% of the control (HCC70, 110±8%). This increase in cell growth was completely prevented in both TNBC cell lines after GPR30 expression was downregulated by treatment with 200 nM gefitinib. In HCC1806 cells, activation of c-src was increased by 17β-estradiol to 350±50% (p<0.01), and gefitinib reduced src activation to 110%. Similar results were obtained in the HCC70 cells. Phosphorylation of EGFR increased to 240±40% (p<0.05) in the HCC1806 cells treated with 17β-estradiol (HCC70, 147±25%). Gefitinib completely prevented this activation. Phosphorylation of CREB and induction of c-fos, cyclin D1 and aromatase expression by 17β-estradiol were all prevented by gefitinib. These experiments conclusively show that reduction of GPER expression is a promising therapeutic approach for TNBC.

  18. Origin of estradiol fatty acid esters in human ovarian follicular fluid.

    Science.gov (United States)

    Pahuja, S L; Kim, A H; Lee, G; Hochberg, R B

    1995-03-01

    The estradiol fatty acid esters are the most potent of the naturally occurring steroidal estrogens. These esters are present predominantly in fat, where they are sequestered until they are hydrolyzed by esterases. Thus they act as a preformed reservoir of estradiol. We have previously shown that ovarian follicular fluid from patients undergoing gonadotropin stimulation contains very high amounts of estradiol fatty acid esters (approximately 10(-7) M). The source of these esters is unknown. They can be formed by esterification of estradiol in the follicular fluid by lecithin:cholesterol acyltransferase (LCAT), or in the ovary by an acyl coenzyme A:acyltransferase. In order to determine which of these enzymatic processes is the source of the estradiol esters in the follicular fluid, we incubated [3H]estradiol with follicular fluid and cells isolated from human ovarian follicular fluid and characterized the fatty acid composition of the [3H]estradiol esters biosynthesized in each. In addition, we characterized the endogenous estradiol fatty acid esters in the follicular fluid and compared them to the biosynthetic esters. The fatty acid composition of the endogenous esters was different than those synthesized by the cellular acyl coenzyme A:acyltransferase, and the same as the esters synthesized by LCAT, demonstrating that the esters are produced in situ in the follicular fluid. Although the role of these estradiol esters in the ovary is not known, given their remarkable estrogenic potency it is highly probable that they have an important physiological role.

  19. Inhibition of cyclooxygenase-2 impacts chondrocyte hypertrophic differentiation during endochondral ossification

    Directory of Open Access Journals (Sweden)

    TJM Welting

    2011-12-01

    Full Text Available Skeletogenesis and bone fracture healing involve endochondral ossification, a process during which cartilaginous primordia are gradually replaced by bone tissue. In line with a role for cyclooxygenase-2 (COX-2 in the endochondral ossification process, non-steroidal anti-inflammatory drugs (NSAIDs were reported to negatively affect bone fracture healing due to impaired osteogenesis. However, a role for COX-2 activity in the chondrogenic phase of endochondral ossification has not been addressed before. We show that COX-2 activity fulfils an important regulatory function in chondrocyte hypertrophic differentiation. Our data reveal essential cross-talk between COX-2 and bone morphogenic protein-2 (BMP-2 during chondrocyte hypertrophic differentiation. BMP-2 mediated chondrocyte hypertrophy is associated with increased COX-2 expression and pharmacological inhibition of COX-2 activity by NSAIDs (e.g., Celecoxib decreases hypertrophic differentiation in various chondrogenic models in vitro and in vivo, while leaving early chondrogenic development unaltered. Our findings demonstrate that COX-2 activity is a novel factor partaking in chondrocyte hypertrophy in the context of endochondral ossification and these observations provide a novel etiological perspective on the adverse effects of NSAIDs on bone fracture healing and have important implications for the use of NSAIDs during endochondral skeletal development.

  20. The effect of magnetic stimulation on the osteogenic and chondrogenic differentiation of human stem cells derived from the adipose tissue (hASCs)

    Science.gov (United States)

    Lima, João; Gonçalves, Ana I.; Rodrigues, Márcia T.; Reis, Rui L.; Gomes, Manuela E.

    2015-11-01

    The use of magnetic nanoparticles (MNPs) towards the musculoskeletal tissues has been the focus of many studies, regarding MNPs ability to promote and direct cellular stimulation and orient tissue responses. This is thought to be mainly achieved by mechano-responsive pathways, which can induce changes in cell behavior, including the processes of proliferation and differentiation, in response to external mechanical stimuli. Thus, the application of MNP-based strategies in tissue engineering may hold potential to propose novel solutions for cell therapy on bone and cartilage strategies to accomplish tissue regeneration. The present work aims at studying the influence of MNPs on the osteogenic and chondrogenic differentiation of human adipose derived stem cells (hASCs). MNPs were incorporated in hASCs and cultured in medium supplemented for osteogenic and chondrogenic differentiation. Cultures were maintained up to 28 days with/without an external magnetic stimulus provided by a magnetic bioreactor, to determine if the MNPs alone could affect the osteogenic or chondrogenic phenotype of the hASCs. Results indicate that the incorporation of MNPs does not negatively affect the viability nor the proliferation of hASCs. Furthermore, Alizarin Red staining evidences an enhancement in extracellular (ECM) mineralization under the influence of an external magnetic field. Although not as evident as for osteogenic differentiation, Toluidine blue and Safranin-O stainings also suggest the presence of a cartilage-like ECM with glycosaminoglycans and proteoglycans under the magnetic stimulus provided. Thus, MNPs incorporated in hASCs under the influence of an external magnetic field have the potential to induce differentiation towards the osteogenic and chondrogenic lineages.

  1. Estradiol replacement enhances fear memory formation, impairs extinction and reduces COMT expression levels in the hippocampus of ovariectomized female mice.

    Science.gov (United States)

    McDermott, Carmel M; Liu, Dan; Ade, Catherine; Schrader, Laura A

    2015-02-01

    Females experience depression, posttraumatic stress disorder (PTSD), and anxiety disorders at approximately twice the rate of males, but the mechanisms underlying this difference remain undefined. The effect of sex hormones on neural substrates presents a possible mechanism. We investigated the effect of ovariectomy at two ages, before puberty and in adulthood, and 17β-estradiol (E2) replacement administered chronically in drinking water on anxiety level, fear memory formation, and extinction. Based on previous studies, we hypothesized that estradiol replacement would impair fear memory formation and enhance extinction rate. Females, age 4 weeks and 10 weeks, were divided randomly into 4 groups; sham surgery, OVX, OVX+low E2 (200nM), and OVX+high E2 (1000nM). Chronic treatment with high levels of E2 significantly increased anxiety levels measured in the elevated plus maze. In both age groups, high levels of E2 significantly increased contextual fear memory but had no effect on cued fear memory. In addition, high E2 decreased the rate of extinction in both ages. Finally, catechol-O-methyltransferase (COMT) is important for regulation of catecholamine levels, which play a role in fear memory formation and extinction. COMT expression in the hippocampus was significantly reduced by high E2 replacement, implying increased catecholamine levels in the hippocampus of high E2 mice. These results suggest that estradiol enhanced fear memory formation, and inhibited fear memory extinction, possibly stabilizing the fear memory in female mice. This study has implications for a neurobiological mechanism for PTSD and anxiety disorders. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. rFN/Cad-11-Modified Collagen Type II Biomimetic Interface Promotes the Adhesion and Chondrogenic Differentiation of Mesenchymal Stem Cells

    Science.gov (United States)

    Guo, Hongfeng; Zhang, Yuan; Li, Zhengsheng; Kang, Fei; Yang, Bo; Kang, Xia; Wen, Can; Yan, Yanfei; Jiang, Bo; Fan, Yujiang

    2013-01-01

    Properties of the cell-material interface are determining factors in the successful function of cells for cartilage tissue engineering. Currently, cell adhesion is commonly promoted through the use of polypeptides; however, due to their lack of complementary or modulatory domains, polypeptides must be modified to improve their ability to promote adhesion. In this study, we utilized the principle of matrix-based biomimetic modification and a recombinant protein, which spans fragments 7–10 of fibronectin module III (heterophilic motif ) and extracellular domains 1–2 of cadherin-11 (rFN/Cad-11) (homophilic motif ), to modify the interface of collagen type II (Col II) sponges. We showed that the designed material was able to stimulate cell proliferation and promote better chondrogenic differentiation of rabbit mesenchymal stem cells (MSCs) in vitro than both the FN modified surfaces and the negative control. Further, the Col II/rFN/Cad-11-MSCs composite stimulated cartilage formation in vivo; the chondrogenic effect of Col II alone was much less significant. These results suggested that the rFN/Cad-11-modified collagen type II biomimetic interface has dual biological functions of promoting adhesion and stimulating chondrogenic differentiation. This substance, thus, may serve as an ideal scaffold material for cartilage tissue engineering, enhancing repair of injured cartilage in vivo. PMID:23919505

  3. Proliferation and chondrogenic differentiation of CD105-positive enriched rat synovium-derived mesenchymal stem cells in three-dimensional porous scaffolds

    International Nuclear Information System (INIS)

    Qi Jun; Chen Anmin; You Hongbo; Li Kunpeng; Zhang Di; Guo Fengjing

    2011-01-01

    Stem cell-based tissue engineering has provided an alternative strategy to treat cartilage lesions, and synovium-derived mesenchymal stem cells (SMSCs) are considered as a promising cell source for cartilage repair. In this study, the SMSCs were isolated from rat synovium, and CD105-positive (CD105 + ) cells were enriched using magnetic activated cell sorting. Sorted cells were subsequently seeded onto the chitosan-alginate composite three-dimensional (3D) porous scaffolds and cultured in chondrogenic culture medium in the presence of TGF-β 3 and BMP-2 for 2 weeks in vitro. After 2 weeks in culture, scanning electron microscopy results showed that cells attached and proliferated well on scaffolds, and secreted extracellular matrix were also observed. From day 7 to day 14, the total DNA and glucosaminoglycan content of the cells cultured in scaffolds were found to have increased significantly, and cell cycle analyses revealed that the percentage of cells in the S and G2/M phases increased and the percentage of cells in the G0/G1 phase decreased. Compared with non-sorted cells, the sorted cells cultured in scaffolds underwent more chondrogenic differentiation, as evidenced by higher expression of type II collagen and Sox9 at the protein and mRNA levels. The results suggest that CD105 + enriched SMSCs may be a potential cell source for cartilage tissue engineering, and the chitosan-alginate composite 3D porous scaffold could provide a favorable microenvironment for supporting proliferation and chondrogenic differentiation of cells.

  4. Anatomical region-dependent enhancement of 3-dimensional chondrogenic differentiation of human mesenchymal stem cells by soluble meniscus extracellular matrix.

    Science.gov (United States)

    Rothrauff, Benjamin B; Shimomura, Kazunori; Gottardi, Riccardo; Alexander, Peter G; Tuan, Rocky S

    2017-02-01

    Extracellular matrix (ECM) derived from decellularized tissues has been found to promote tissue neogenesis, most likely mediated by specific biochemical and physical signaling motifs that promote tissue-specific differentiation of progenitor cells. Decellularized ECM has been suggested to be efficacious for the repair of tissue injuries. However, decellularized meniscus contains a dense collagenous structure, which impedes cell seeding and infiltration and is not readily applicable for meniscus repair. In addition, the meniscus consists of two distinct anatomical regions that differ in vascularity and cellular phenotype. The purpose of this study was to explore the region-specific bioactivity of solubilized ECM derived from the inner and outer meniscal regions as determined in 2D and 3D cultures of adult mesenchymal stem cells (MSCs). When added as a medium supplement to 2D cultures of MSCs, urea-extracted fractions of the inner (imECM) and outer meniscal ECM (omECM) enhanced cell proliferation while imECM most strongly upregulated fibrochondrogenic differentiation on the basis of gene expression profiles. When added to 3D cultures of MSCs seeded in photocrosslinked methacrylated gelatin (GelMA) hydrogels, both ECM fractions upregulated chondrogenic differentiation as determined by gene expression and protein analyses, as well as elevated sulfated glycosaminoglycan sGAG content, compared to ECM-free controls. The chondrogenic effect at day 21 was most pronounced with imECM supplementation, but equivalent between ECM groups by day 42. Despite increased cartilage matrix, imECM and omECM constructs possessed compressive moduli similar to controls. In conclusion, soluble meniscal ECM may be considered for use as a tissue-specific reagent to enhance chondrogenesis for MSC-based 3D cartilage tissue engineering. The inner region of the knee meniscus is frequently injured and possesses a poor intrinsic healing capacity. Solubilized extracellular matrix (ECM) derived from

  5. In vitro bioactivity of 17alpha-estradiol.

    Science.gov (United States)

    Sievernich, André; Wildt, Ludwig; Lichtenberg-Fraté, Hella

    2004-12-01

    A miniaturised short-term in vitro assay based on the activation of the human estrogen receptor alpha and genetically modified yeast (Saccharomyces cerevisiae) cells was performed to explore the capacity of this system to monitor the bioactivity of estrogenic compounds, particularly 17alpha- and 17beta-estradiol. Together with the human estrogen receptor (hER)-alpha plasmid, the reporter plasmid containing a yeast-optimised version of the green fluorescent protein (yEGFP) linked to three repeats of the cis-acting estrogen hormone-responsive element (ERE) were expressed in a strain being deleted in the pleiotropic drug resistance transporters Pdr5, Snq2 and Yor1, known to facilitate efflux of organic compounds including steroids and chemotherapeutics. Agonists that bind to hER in vitro trigger estrogen receptor-mediated transcriptional activation of the GFP reporter gene monitored by fluorescence emission at 535 nm. The sensitivity of the assay was tested with various 17alpha- and 17beta-estradiol concentrations, yielding a detection limit of 5 pg/ml (0.018 nM) for the agonist 17beta-E2 in solvent and in human charcoal-stripped serum using a S. cerevisiae pdr5, snq2 and yor1 mutant strain. For 17alpha-estradiol only, at approximately 1500 pg/ml a similar fluorescence response compared to 100 pg/ml 17beta-E2 was observed implicating a much weaker potency of this stereoisomer. The specificity of the system was tested by expression of a truncated hER lacking the ligand-binding domain E and by administration of the androgen, 4-androsten 3,17 dione. Both controls did not yield an increase in fluorescence emission. This fluorescence emission assay enables detection of estrogenic biological activity induced by direct agonists, such as 17beta-E2 at concentrations similar to those found in human sera or by estrogen-like chemicals.

  6. Methylation of the chicken vitellogenin gene: influence of estradiol administration.

    Science.gov (United States)

    Meijlink, F C; Philipsen, J N; Gruber, M; Ab, G

    1983-01-01

    The degree of methylation of the chicken vitellogenin gene has been investigated. Upon induction by administration of estradiol to a rooster, methyl groups at specific sites near the 5'-end of the gene are eliminated. The process of demethylation is slower than the activation of the gene. Demethylation is therefore probably not a prerequisite to gene transcription. At least two other sites in the coding region of the gene are methylated in the liver of estrogenized roosters, but not in the liver of a laying hen, where the gene is naturally active. Images PMID:6298743

  7. Estradiol increases the sensitivity of ventral tegmental area dopamine neurons to dopamine and ethanol.

    Directory of Open Access Journals (Sweden)

    Bertha J Vandegrift

    Full Text Available Gender differences in psychiatric disorders such as addiction may be modulated by the steroid hormone estrogen. For instance, 17β-estradiol (E2, the predominant form of circulating estrogen in pre-menopausal females, increases ethanol consumption, suggesting that E2 may affect the rewarding properties of ethanol and thus the development of alcohol use disorder in females. The ventral tegmental area (VTA is critically involved in the rewarding and reinforcing effects of ethanol. In order to determine the role of E2 in VTA physiology, gonadally intact female mice were sacrificed during diestrus II (high E2 or estrus (low E2 for electrophysiology recordings. We measured the excitation by ethanol and inhibition by dopamine (DA of VTA DA neurons and found that both excitation by ethanol and inhibition by dopamine were greater in diestrus II compared with estrus. Treatment of VTA slices from mice in diestrus II with an estrogen receptor antagonist (ICI 182,780 reduced ethanol-stimulated neuronal firing, but had no effect on ethanol-stimulated firing of neurons in slices from mice in estrus. Surprisingly, ICI 182,780 did not affect the inhibition by DA, indicating different mechanisms of action of estrogen receptors in altering ethanol and DA responses. We also examined the responses of VTA DA neurons to ethanol and DA in ovariectomized mice treated with E2 and found that E2 treatment enhanced the responses to ethanol and DA in a manner similar to what we observed in mice in diestrus II. Our data indicate that E2 modulates VTA neuron physiology, which may contribute to both the enhanced reinforcing and rewarding effects of alcohol and the development of other psychiatric disorders in females that involve alterations in DA neurotransmission.

  8. In Vivo Evaluation of Biocompatibility and Chondrogenic Potential of a Cell-Free Collagen-Based Scaffold

    Directory of Open Access Journals (Sweden)

    Giovanna Calabrese

    2017-11-01

    Full Text Available Injured articular cartilage has a limited innate regenerative capacity, due to the avascular nature and low cellularity of the tissue itself. Although several approaches have been proposed to repair the joint cartilage, none of them has proven to be effective. The absence of suitable therapeutic options has encouraged tissue-engineering approaches combining specific cell types and biomaterials. In the present work, we have evaluated the potential of a cell-free Collagen I-based scaffold to promote the augmentation of cartilage-like phenotype after subcutaneous implantation in the mouse. Forty female mice were grafted subcutaneously with scaffolds, while four additional mice without scaffold were used as negative controls. The effects of scaffold were evaluated at 1, 2, 4, 8, or 16 weeks after implantation. Immunohistochemical analysis shows the expression of typical cartilage markers, including type-II Collagen, Aggrecan, Matrilin-1 and Sox 9. These data are also confirmed by qRT-PCR that further show that both COL2A1 and COL1A1 increase over time, but the first one increases more rapidly, thus suggesting a typical cartilage-like address. Histological analysis shows the presence of some pericellular lacunae, after 8 and 16 weeks. Results suggest that this scaffold (i is biocompatible in vivo, (ii is able to recruit host cells (iii induce chondrogenic differentiation of host cells. Such evidences suggest that this cell-free scaffold is promising and represents a potential approach for cartilage regeneration.

  9. Induction of Chondrogenic Differentiation of Human Mesenchymal Stem Cells by Biomimetic Gold Nanoparticles with Tunable RGD Density.

    Science.gov (United States)

    Li, Jingchao; Li, Xiaomeng; Zhang, Jing; Kawazoe, Naoki; Chen, Guoping

    2017-07-01

    Nanostructured materials have drawn a broad attention for their applications in biomedical fields. Ligand-modified nanomaterials can well mimic the dynamic extracellular matrix (ECM) microenvironments to regulate cell functions and fates. Herein, ECM mimetic gold nanoparticles (Au NPs) with tunable surface arginine-glycine-aspartate (RGD) density are designed and synthesized to induce the chondrogenic differentiation of human mesenchymal stem cells (hMSCs). The biomimetic Au NPs with an average size of 40 nm shows good biocompatibility without affecting the cell proliferation in the studied concentration range. The RGD motifs on Au NPs surface facilitate cellular uptake of NPs into monolayer hMSCs through integrin-mediated endocytosis. The biomimetic NPs have a promotive effect on cartilaginous matrix production and marker gene expression in cell pellet culture, especially for the biomimetic Au NPs with high surface RGD density. This study provides a novel strategy for fabricating biomimetic NPs to regulate cell differentiation, which holds great potentials in tissue engineering and biomedical applications. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Use of the second harmonic generation microscopy to evaluate chondrogenic differentiation of mesenchymal stem cells for cartilage repair

    Science.gov (United States)

    Bordeaux-Rego, P.; Baratti, M. O.; Duarte, A. S. S.; Ribeiro, T. B.; Andreoli-Risso, M. F.; Vidal, B.; Miranda, J. B.; Adur, J.; de Thomaz, A. A.; Pelegati, V. B.; Costa, F. F.; Carvalho, H. F.; Cesar, C. L.; Luzo, A.; Olalla Saad, S. T.

    2012-03-01

    Articular cartilage injury remains one of the major concerns in orthopedic surgery. Mesenchymal stem cell (MSC) transplantation has been introduced to avoid some of the side effects and complications of current techniques.. With the aim to evaluate chondrogenic differentiation of mesenchymal stem cells, we used Second Harmonic Generation (SHG) microscopy to analyze the aggregation and orientation of collagen fibrils in the hyaline cartilage of rabbit knees. The experiment was performed using implants with type II collagen hydrogel (a biomaterial that mimics the microenvironment of the cartilage), one implant containing MSC and one other without MSC (control). After 10 weeks, the rabbit knees were dissected and fibril collagen distribution and spatial organization in the extracellular matrix of the lesions were verified by SHG. The result showed significant differences, whereas in histological sections of the cartilaginous lesions with MSC the collagen fibers are organized and regular; in the control sections the collagen fibers are more irregular, with absence of cells. A macroscopic analysis of the lesions confirmed this difference, showing a greater percentage of lesions filling in knees treated with MSC than in the knees used as controls. This study demonstrates that SHG microscopy will be an excellent tool to help in the evaluation of the effectiveness of MSC-based cell therapy for cartilage repair.

  11. Estradiol suppresses ingestive response evoked by activation of 5-HT1A receptors in the lateral hypothalamus of ovariectomized rats.

    Science.gov (United States)

    Taschetto, Ana P D; Levone, Brunno R; Kochenborger, Larissa; da Silva, Eduardo S; Flores, Rafael A; Faria, Moacir S; Paschoalini, Marta A

    2018-03-08

    The present study investigated the effects of estradiol (E2) on ingestive behavior after activation of 5-HT1A receptors in the lateral hypothalamus (LH) of female rats habituated to eat a wet mash diet. Ovariectomized rats treated with corn oil (OVX) or estradiol cypionate (OVX+E) received local injections into the LH of vehicle or an agonist of 5-HT1A receptors, 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT; at a dose of 6 nmol). To determine the involvement of these receptors in food intake, some animals were pretreated with N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane carboxamide maleate (WAY-100635, a 5-HT1A receptor full antagonist, at a dose of 0.37 nmol), followed by the injection of the agonist 8-OH-DPAT or its vehicle. The results showed that the injection of 8-OH-DPAT into the LH of OVX rats significantly increased food intake, and the duration and frequency of this behavior. The pretreatment with E2 suppressed the hyperphagic response induced by 8-OH-DPAT in OVX animals. The inhibition of 5-HT1A receptors after pretreatment with WAY-100635 blocked the hyperphagic effects evoked by 8-OH-DPAT in OVX. These results indicate that the activity of LH 5-HT1A receptors could be affected by blood E2 levels.

  12. Modulation of 17{beta}-estradiol-induced responses in fish by cytochrome P4501A1 inducing compounds

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, M.J.; Hinton, D.E. [Univ. of California, Davis, CA (United States)

    1995-12-31

    Some compounds which induce cytochrome P4501A1 (CYP1A1) are antiestrogenic in mammalian bioassay, and this effect is linked to aryl hydrocarbon (Ah) receptor. Liver of fish synthesizes estrogen-inducible egg yolk precursor protein vitellogenin (Vg) which is critical for oocyte maturation and ovarian development. To determine if Ah receptor-linked endocrine modulation could occur in fish liver, primary cultures of juvenile rainbow trout (Oncorhynchus mykiss) liver cells were co-administered 17{beta}-estradiol and CYP1A1 inducing compounds. Vitellogenin and albumin, estimated by ELISA measurement of concentration in the media 48 hrs after treatment, formed the basis for the test. Cellular CYP1A1 protein content and catalytic activity was estimated by ELISA and ethoxyresorufin-O-deethylase (EROD) activity assays respectively. Equivalent viability (mitochondrial dehydrogenase activity) and secretary functional capacity (albumin synthesis) were estimated and correlated with other results. In descending order, 2,3,4,7,8 pentachlorodibenzofuran (10{sup {minus}12} to 10{sup {minus}8} M) > 2,3,7,8 tetrachlorodibenzo-p-dioxin {approx_equal} 2,3,7,8 tetrachlorodibenzofuran (10{sup {minus}11} to 10{sup {minus}8} M) > {beta}-naphthoflavone (10{sup {minus}7} to 10{sup {minus}6} M) inhibited Vg synthesis in 17{beta}-estradiol treated liver cells. Potency of inhibition directly related to strength as an inducer of CYP1A1 protein. At 10-8 M, PCB congeners 77, 126, and 156 did not inhibit Vg synthesis and induced no or only moderate CYP1A1 protein. At 10-8 M, PCB congener 114, a weak CYP1A1 inducer, potentiated Vg synthesis relative to cells treated with 17{beta}-estradiol alone. This study increases their understanding of the consequences of hepatic CYP1A1 induction, forewarns of reproductive impairment of sexually maturing fishes exposed to CYP1A1 inducing compounds and argues for further, more detailed in vivo investigation.

  13. Analogues of estradiol as potential breast tumor imaging agents

    International Nuclear Information System (INIS)

    Gibson, R.E.; Rzeszotarski, W.J.; Ferriera, N.L.; Jagoda, E.M.; Reba, R.C.; Eckelman, W.C.

    1984-01-01

    The radioiodinated analogue of estradiol, 11β-methoxy-17α-[/sup 125/I]iodovinylestradiol (MIVE/sub 2/), has been shown to be a good candidate for the imaging of estrogen dependent breast tumors. Although there has been no extensive study on the sensitivity of radiotracers of this type, the authors have not observed localization of the radiotracer in metastatic lesions containing less than 20 fmole estrogen receptor/mg protein or in bone metasteses. In order to improve the sensitivity, they have examined several structural analogues of moxestrol (the parent structure for MIVE/sub 2/) for affinity to the ER isolated from immature rat uterus. The 11β-ethyl analogue (EEE/sub 2/) of ethynyl estradiol (EE/sub 2/) exhibits the highest affinity with the 11β-methyl analogue second best. Although the lipophilicity is also very high this compound should not be much more lipophilic than 16-iodoestradiol or MIVE/sub 2/ since the introduction of iodine increases the log P by greater than 1. The distribution of the tritiated derivative of EEE/sub 2/ is under study

  14. An improved method for estradiol-17B radioimmunoassay

    International Nuclear Information System (INIS)

    El-Banna, I.M.; El-Asrag, H.A.; Gamal, M.H.

    1991-01-01

    This work describes an improved radioimmunoassay (RIA) of serum estradiol-17 B (E) using locally generated immuno-chemicals. Estradiol hemisuccinate (E -3-H S) was prepared and conjugated to bovine serum albumin (BSA). The obtained conjugate; E 3-H S: BSA, hadλ max at 280 mu and the steroid BSA molar ratio was 25:1. The immunogen was injected subcutaneously in New Zealand rabbits and large amount of antiserum was harvested with 1 : 10500 antibody titre. The antibody cross reactions with estrone (E ), estriol (E ) and progesterone (P) were determined. Blood samples were collected from cycling Osemi ewes during follicular phase, pregnant ewes near term and daily from a cycling ewe over two consecutive estrous cycles. Serum samples were analysed for E both directly and after diethyl ether extraction (DE). The higher E values were found in the direct assay for pregnant ewes. The direct serum minnature RIA system, described herein, was found to be specific, sensitive, precise and economic.5 fig. 2 tab

  15. Direct radioimmunoassay of 17. beta. -estradiol in ether extracts of bovine

    Energy Technology Data Exchange (ETDEWEB)

    Medina, M.B.

    Anabolic estrogens such as 17..beta..-estradiol or 17..beta..-estradiol benzoate are used to promote growth and increase feed efficiency in food-producing cattle. This paper describes a technique to produce a more specific antibody to 17..beta..-estradiol by intradermal immunization using microquantities of 6-(carboxymethyl)-17..beta..-estradiol oxime bovine serum albumin and the development of a radioimmunoassay (RIA) procedure to measure directly the amounts of 17..beta..-estradiol in ether extracts of bovine serum without using cleanup procedures. Results demonstrated that a specific and sensitive antibody was produced, and a titer of 1:10,000 was used in the RIA procedure. Antibody cross-reactivity with ..beta..-estradiol metabolites and other anabolic estrogens was negligible. The untreated bovine sera showed 0-24 pg of apparent 17..beta..-estradiol/mL, while 0-31 pg/mL total estrogens had been reported in the literature. This assay can measure 5-100 pg in 20-250..mu..L/sample. This method can be used before or immediately after slaughter to monitor the residual amounts of estradiol used in the treatment of cattle.

  16. Direct radioimmunoassay of 17β-estradiol in ether extracts of bovine

    International Nuclear Information System (INIS)

    Medina, M.B.

    1986-01-01

    Anabolic estrogens such as 17β-estradiol or 17β-estradiol benzoate are used to promote growth and increase feed efficiency in food-producing cattle. This paper describes a technique to produce a more specific antibody to 17β-estradiol by intradermal immunization using microquantities of 6-(carboxymethyl)-17β-estradiol oxime bovine serum albumin and the development of a radioimmunoassay (RIA) procedure to measure directly the amounts of 17β-estradiol in ether extracts of bovine serum without using cleanup procedures. Results demonstrated that a specific and sensitive antibody was produced, and a titer of 1:10,000 was used in the RIA procedure. Antibody cross-reactivity with β-estradiol metabolites and other anabolic estrogens was negligible. The untreated bovine sera showed 0-24 pg of apparent 17β-estradiol/mL, while 0-31 pg/mL total estrogens had been reported in the literature. This assay can measure 5-100 pg in 20-250μL/sample. This method can be used before or immediately after slaughter to monitor the residual amounts of estradiol used in the treatment of cattle

  17. Viral Vector Mediated Over-Expression of Estrogen Receptor–α in Striatum Enhances the Estradiol-induced Motor Activity in Female Rats and Estradiol Modulated GABA Release

    Science.gov (United States)

    Schultz, Kristin N.; von Esenwein, Silke A.; Hu, Ming; Bennett, Amy L.; Kennedy, Robert T.; Musatov, Sergei; Toran-Allerand, C. Dominique; Kaplitt, Michael G.; Young, Larry J.; Becker, Jill B.

    2009-01-01

    Classical estrogen receptor signaling mechanisms involve estradiol binding to intracellular nuclear receptors (estrogen receptor-α (ERα) and estrogen receptor-β (ERβ)) to promote changes in protein expression. Estradiol can also exert effects within seconds to minutes, however, a timescale incongruent with genomic signaling. In the brain, estradiol rapidly potentiates stimulated dopamine release in the striatum of female rats and enhances spontaneous rotational behavior. Furthermore, estradiol rapidly attenuates the K+- evoked increase of GABA in dialysate. We hypothesize that these rapid effects of estradiol in the striatum are mediated by ERα located on the membrane of medium spiny GABAergic neurons. This experiment examined whether over-expression of ERα in the striatum would enhance the effect of estradiol on rotational behavior and the K+- evoked increase in GABA in dialysate. Ovariectomized female rats were tested for rotational behavior or underwent microdialysis experiments after unilateral intrastriatal injections of a recombinant adeno-associated virus (AAV) containing the human ERα cDNA (AAV.ERα) into the striatum; controls received either the same vector into areas outside the striatum or an AAV containing the human alkaline phosphatase gene into the striatum (AAV.ALP). Animals that received AAV.ERα in the striatum exhibited significantly greater estradiol-induced contralateral rotations compared to controls and exhibited behavioral sensitization of contralateral rotations induced by a low dose of amphetamine. ERα over-expression also enhanced the inhibitory effect of estradiol on K+- evoked GABA release suggesting that disinhibition of dopamine release from terminals in the striatum resulted in the enhanced rotational behavior. PMID:19211896

  18. Viral vector-mediated overexpression of estrogen receptor-alpha in striatum enhances the estradiol-induced motor activity in female rats and estradiol-modulated GABA release.

    Science.gov (United States)

    Schultz, Kristin N; von Esenwein, Silke A; Hu, Ming; Bennett, Amy L; Kennedy, Robert T; Musatov, Sergei; Toran-Allerand, C Dominique; Kaplitt, Michael G; Young, Larry J; Becker, Jill B

    2009-02-11

    Classical estrogen receptor-signaling mechanisms involve estradiol binding to intracellular nuclear receptors [estrogen receptor-alpha (ERalpha) and estrogen receptor-beta (ERbeta)] to promote changes in protein expression. Estradiol can also exert effects within seconds to minutes, however, a timescale incongruent with genomic signaling. In the brain, estradiol rapidly potentiates stimulated dopamine release in the striatum of female rats and enhances spontaneous rotational behavior. Furthermore, estradiol rapidly attenuates the K(+)-evoked increase of GABA in dialysate. We hypothesize that these rapid effects of estradiol in the striatum are mediated by ERalpha located on the membrane of medium spiny GABAergic neurons. This experiment examined whether overexpression of ERalpha in the striatum would enhance the effect of estradiol on rotational behavior and the K(+)-evoked increase in GABA in dialysate. Ovariectomized female rats were tested for rotational behavior or underwent microdialysis experiments after unilateral intrastriatal injections of a recombinant adeno-associated virus (AAV) containing the human ERalpha cDNA (AAV.ERalpha) into the striatum; controls received either the same vector into areas outside the striatum or an AAV containing the human alkaline phosphatase gene into the striatum (AAV.ALP). Animals that received AAV.ERalpha in the striatum exhibited significantly greater estradiol-induced contralateral rotations compared with controls and exhibited behavioral sensitization of contralateral rotations induced by a low-dose of amphetamine. ERalpha overexpression also enhanced the inhibitory effect of estradiol on K(+)-evoked GABA release suggesting that disinhibition of dopamine release from terminals in the striatum resulted in the enhanced rotational behavior.

  19. Estradiol-promoted accumulation of receptor in nuclei of porcine endometrium cells. Immunogold electron microscopy of resting and estradiol-stimulated cells.

    Science.gov (United States)

    Sierralta, W D; Jakob, F; Thole, H; Engel, P; Jungblut, P W

    1992-01-01

    Endometrium was collected by curettage from castrated pigs, either untreated or exposed to estradiol in vivo by intrauterine injection, and processed for electron microscopy. The resin LR Gold was used for embedding, and sections were floated on droplets of 10 nm diameter gold particles, coated with the immunoglobulin-G1 (IgG1) fraction or its Fab2 fragment of a monospecific polyclonal antiserum raised in goats against the C-terminal half of the estradiol receptor. On average, only one gold particle per microns 2 became attached in the cytoplasmic area of untreated cells, whereas four were found over the nuclear area. These figures rose to 2-3/microns 2 and 15-26/microns 2, respectively, within 10 min after exposure to estradiol. The labeling intensities of nuclei in cell clusters and of coprocessed nuclei released from cells ruptured during curettage were identical in all situations. Nuclear pores were frequently tagged after estradiol treatment. The proportions of tagging densities in nuclei of untreated and estradiol-exposed cells corresponded to those of receptor contents measured in extracts of isolated nuclei by ligand binding. This correlation was not seen for the cytoplasmic compartment of untreated cells, the scarce tagging of which is interpreted by hidden antigenic determinants. Our morphological analyses support the conclusions drawn from biochemical data (Sierralta et al., 1992) of an estradiol-promoted translocation of receptor from the cytoplasm into the nucleus.

  20. Estradiol Synthesis in Gut-Associated Lymphoid Tissue: Leukocyte Regulation by a Sexually Monomorphic System.

    Science.gov (United States)

    Oakley, Oliver R; Kim, Kee Jun; Lin, Po-Ching; Barakat, Radwa; Cacioppo, Joseph A; Li, Zhong; Whitaker, Alexandra; Chung, Kwang Chul; Mei, Wenyan; Ko, CheMyong

    2016-12-01

    17β-estradiol is a potent sex hormone synthesized primarily by gonads in females and males that regulates development and function of the reproductive system. Recent studies show that 17β-estradiol is locally synthesized in nonreproductive tissues and regulates a myriad of events, including local inflammatory responses. In this study, we report that mesenteric lymph nodes (mLNs) and Peyer's patches (Pps) are novel sites of de novo synthesis of 17β-estradiol. These secondary lymphoid organs are located within or close to the gastrointestinal tract, contain leukocytes, and function at the forefront of immune surveillance. 17β-estradiol synthesis was initially identified using a transgenic mouse with red fluorescent protein coexpressed in cells that express aromatase, the enzyme responsible for 17β-estradiol synthesis. Subsequent immunohistochemistry and tissue culture experiments revealed that aromatase expression was localized to high endothelial venules of these lymphoid organs, and these high endothelial venule cells synthesized 17β-estradiol when isolated and cultured in vitro. Both mLNs and Pps contained 17β-estradiol with concentrations that were significantly higher than those of peripheral blood. Furthermore, the total amount of 17β-estradiol in these organs exceeded that of the gonads. Mice lacking either aromatase or estrogen receptor-β had hypertrophic Pps and mLNs with more leukocytes than their wild-type littermates, demonstrating a role for 17β-estradiol in leukocyte regulation. Importantly, we did not observe any sex-dependent differences in aromatase expression, 17β-estradiol content, or steroidogenic capacity in these lymphoid organs.

  1. Effect of estradiol on radiation-induced chromosome aberrations in human lymphocytes

    International Nuclear Information System (INIS)

    Kanda, Reiko; Hayata, Isamu

    1999-01-01

    As a part of studies on physiological factors that affect radiosensitivity, we examined the in vitro effect of estradiol (E2) on the yield of radiation-induced chromosome aberrations in human peripheral lymphocytes. Lymphocytes were cultured for 3 days in the medium containing E2 at 0-100000 ng/ml. On the second day, they were irradiated by X-rays at 3 Gy, and then 2% phytohemagglutinin and 0.05 μg/ml colcemid were added to the medium. After further 48 h, mitotic indices and the yields of chromosome aberrations were examined at various E2 concentrations. E2 treatment at concentrations above 1000 ng/ml resulted in dose-related inhibition of mitosis. Repeated experiments showed that the yield of dicentrics plus centric rings in the culture containing E2 at 100 ng/ml was significantly higher than the yields at 0 ng/ml. Similarly, the yield of total chromosome breaks in the culture containing E2 at 100 ng/ml was significantly higher than that at 1 ng/ml. This study provides the direct evidence in human that radiosensitivity may vary in relation to hormonal conditions. (author)

  2. Differential effects of estradiol on carotid artery inflammation when administered early versus late after surgical menopause.

    Science.gov (United States)

    Sophonsritsuk, Areepan; Appt, Susan E; Clarkson, Thomas B; Shively, Carol A; Espeland, Mark A; Register, Thomas C

    2013-05-01

    The aim of this study was to determine the effects of estrogen therapy (ET) on carotid artery inflammation when initiated early and late relative to surgical menopause. Female cynomolgus macaques consuming atherogenic diets were ovariectomized and randomized to control or oral estradiol (E2; human equivalent dose of 1 mg/d micronized E2) initiated at 1 month (early menopause, n = 24) or 54 months (late menopause, n = 40) after ovariectomy. The treatment period was 8 months. Carotid artery expression of the markers of monocyte/macrophages (CD68 and CD163), dendritic cells (CD83), natural killer cells (neural cell adhesion molecule-1), and interferon-γ was significantly lower in E2-treated animals in the early menopause group but not in the late menopause group (P menopausal stage (P menopause inhibits macrophage accumulation in the carotid artery, an effect that is not observed when E2 is administered after several years of estrogen deficiency. No evidence for pro-inflammatory effects of late ET is observed. The results provide support for the timing hypothesis of postmenopausal ET with implications for the interpretation of outcomes in the Women's Health Initiative.

  3. Estradiol-Dependent Stimulation and Suppression of Gonadotropin-Releasing Hormone Neuron Firing Activity by Corticotropin-Releasing Hormone in Female Mice.

    Science.gov (United States)

    Phumsatitpong, Chayarndorn; Moenter, Suzanne M

    2018-01-01

    Gonadotropin-releasing hormone (GnRH) neurons are the final central regulators of reproduction, integrating various inputs that modulate fertility. Stress typically inhibits reproduction but can be stimulatory; stress effects can also be modulated by steroid milieu. Corticotropin-releasing hormone (CRH) released during the stress response may suppress reproduction independent of downstream glucocorticoids. We hypothesized CRH suppresses fertility by decreasing GnRH neuron firing activity. To test this, mice were ovariectomized (OVX) and either implanted with an estradiol capsule (OVX+E) or not treated further to examine the influence of estradiol on GnRH neuron response to CRH. Targeted extracellular recordings were used to record firing activity from green fluorescent protein-identified GnRH neurons in brain slices before and during CRH treatment; recordings were done in the afternoon when estradiol has a positive feedback effect to increase GnRH neuron firing. In OVX mice, CRH did not affect the firing rate of GnRH neurons. In contrast, CRH exhibited dose-dependent stimulatory (30 nM) or inhibitory (100 nM) effects on GnRH neuron firing activity in OVX+E mice; both effects were reversible. The dose-dependent effects of CRH appear to result from activation of different receptor populations; a CRH receptor type-1 agonist increased firing activity in GnRH neurons, whereas a CRH receptor type-2 agonist decreased firing activity. CRH and specific agonists also differentially regulated short-term burst frequency and burst properties, including burst duration, spikes/burst, and/or intraburst interval. These results indicate that CRH alters GnRH neuron activity and that estradiol is required for CRH to exert both stimulatory and inhibitory effects on GnRH neurons. Copyright © 2018 Endocrine Society.

  4. Progesterone and estradiol exert an inhibitory effect on the production of anti-inflammatory cytokine IL-10 by activated MZ B cells.

    Science.gov (United States)

    Bommer, I; Muzzio, D O; Zygmunt, M; Jensen, F

    2016-08-01

    The main message of this work is the fact that female sex hormones, progesterone and estradiol, whose levels significantly rise during pregnancy, inhibit the production of anti-inflammatory cytokine IL-10 with no apparent effect on pro-inflammatory cytokine TNF-α by activated MZ B cells. This is an important piece of information and helps to better understand how the maternal immune system controls the balance between immune tolerance and immune activation during pregnancy leading to the simultaneously acceptance of the semi-allogeneic fetus and the proper defense of the mother against pathogens during this critical period of time. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  5. In vitro regulation of LH biosynthesis and release by GnRH and estradiol

    International Nuclear Information System (INIS)

    Ramey, J.W.

    1986-01-01

    Anterior pituitaries were taken from female rats at random stages of the estrous cycle, enzymatically dispersed, and cultured for 48h in steroid-free α-modified Eagles medium followed by 24h in fresh medium +/- estradiol (E 2 ). The pituitary cells were then incubated in fresh medium containing radiolabeled precursors +/- gonadotropin releasing hormone (GnRH). Radioactive precursor incorporation into LH was determined by immuno-precipitation. The dose-dependent effects of E 2 (10 -11 to 10 -8 M) on 3 H-glucosamine ( 3 H-Gln) and 35 S-methionine ( 35 S-Met) incorporation into LH +/- 1 nM GnRH (4h) were investigated. GnRH (10 -9 M) and E 2 (all doses) significantly increased total 3 H-Gln LH. Moreover, E 2 at 10 -9 M and 10 -8 M significantly enhanced GnRH stimulated LH glycosylation. In contrast, addition of GnRH and/or E 2 did not significantly increase 35 S-Met incorporation into LH over a 4h period. The effects of various GnRH concentrations (10 -11 to 10 -9 M; 8h) +/- E 2 (0.05 nM) on 3 H-Gln LH and 35 S-Met LH production were also investigated. In the absence of E 2 , only 10 -9 M GnRH was effective in increasing total 3 H-Gln LH and 35 S-Met LH synthesis. However, in the presence of E 2 , all concentrations of GnRH stimulated LH synthesis with 3 H-Gln LH production responding in a dose related manner whereas 35 S-Met LH production was maximally stimulated at all doses of GnRH. In the final series of experiments, pituitary cells previously exposed to estradiol were incubated for 4 h in normal calcium or low calcium medium containing 3 H-Gln or 35 S-Met +/- GnRH. Removal of extracellular calcium completely inhibited GnRH stimulated 3 H-Gln LH and 35 S-Met LH production

  6. Estradiol Membrane-Initiated Signaling in the Brain Mediates Reproduction.

    Science.gov (United States)

    Micevych, Paul E; Mermelstein, Paul G; Sinchak, Kevin

    2017-11-01

    Over the past few years our understanding of estrogen signaling in the brain has expanded rapidly. Estrogens are synthesized in the periphery and in the brain, acting on multiple receptors to regulate gene transcription, neural function, and behavior. Various estrogen-sensitive signaling pathways often operate in concert within the same cell, increasing the complexity of the system. In females, estrogen concentrations fluctuate over the estrous/menstrual cycle, dynamically modulating estrogen receptor (ER) expression, activity, and trafficking. These dynamic changes influence multiple behaviors but are particularly important for reproduction. Using the female rodent model, we review our current understanding of estradiol signaling in the regulation of sexual receptivity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Loneliness depends on salivary estradiol levels in adolescent females.

    Science.gov (United States)

    Fujisawa, Takashi X; Nishitani, Shota; Obara, Tatsuro; Shinohara, Kazuyuki

    2012-01-01

    Loneliness is one of the psychological characteristics in adolescence, during which sex hormones are elevated. The elevation of sex steroid hormones is known to sculpture and remodel neuronal circuits, which cause behavioral characteristics in adolescence. The aim of the present study is to investigate the relationship between loneliness and sex steroid hormones, testosterone (T) and 17β-estradiol (E2). Fifty-eight adolescents (28 boys and 30 girls) participated in this study. The salivary levels of T and E2 were measured by Enzyme-Linked Immunosorbent Assay (ELISA). Loneliness was assessed by the UCLA loneliness scale, which is widely used as a self-administered questionnaire. The results showed that Salivary E2 levels had positive relevance to loneliness in females, whereas there was no relationship in males. Salivary T level was not shown to be relevant with loneliness in either sex group. These findings suggest that E2 has gender specific effects on loneliness in adolescent females.

  8. Relationship between Carotenoids, Retinol, and Estradiol Levels in Older Women

    Directory of Open Access Journals (Sweden)

    Marcello Maggio

    2015-08-01

    Full Text Available Background. In vitro evidence suggests anti-estrogenic properties for retinol and carotenoids, supporting a chemo-preventive role of these phytochemicals in estrogen-dependent cancers. During aging there are significant reductions in retinol and carotenoid concentrations, whereas estradiol levels decline during menopause and progressively increase from the age of 65. We aimed to investigate the hypothesis of a potential relationship between circulating levels of retinol, carotenoids, and estradiol (E2 in a cohort of late post-menopausal women. Methods. We examined 512 women ≥ 65 years from the InCHIANTI study. Retinol, α-caroten, β-caroten, β-criptoxantin, lutein, zeaxanthin, and lycopene levels were assayed at enrollment (1998–2000 by High-Performance Liquid Chromatography. Estradiol and testosterone (T levels were assessed by Radioimmunometry (RIA and testosterone-to-estradiol ratio (T/E2, as a proxy of aromatase activity, was also calculated. General linear models adjusted for age (Model 1 and further adjusted for other confounders including Body Mass Index (BMI BMI, smoking, intake of energy, lipids, and vitamin A; C-Reactive Protein, insulin, total cholesterol, liver function, and testosterone (Model 2 were used to investigate the relationship between retinol, carotenoids, and E2 levels. To address the independent relationship between carotenoids and E2 levels, factors significantly associated with E2 in Model 2 were also included in a fully adjusted Model 3. Results. After adjustment for age, α-carotene (β ± SE = −0.01 ± 0.004, p = 0.02 and β-carotene (β ± SE = −0.07 ± 0.02, p = 0.0007 were significantly and inversely associated with E2 levels. α-Carotene was also significantly and positively associated with T/E2 ratio (β ± SE = 0.07 ± 0.03, p = 0.01. After adjustment for other confounders (Model 2, the inverse relationship between α-carotene (β ± SE = −1.59 ± 0.61, p = 0.01, β-carotene (β ± SE = −0.29

  9. Relationship between Carotenoids, Retinol, and Estradiol Levels in Older Women.

    Science.gov (United States)

    Maggio, Marcello; de Vita, Francesca; Lauretani, Fulvio; Bandinelli, Stefania; Semba, Richard D; Bartali, Benedetta; Cherubini, Antonio; Cappola, Anne R; Ceda, Gian Paolo; Ferrucci, Luigi

    2015-08-05

    In vitro evidence suggests anti-estrogenic properties for retinol and carotenoids, supporting a chemo-preventive role of these phytochemicals in estrogen-dependent cancers. During aging there are significant reductions in retinol and carotenoid concentrations, whereas estradiol levels decline during menopause and progressively increase from the age of 65. We aimed to investigate the hypothesis of a potential relationship between circulating levels of retinol, carotenoids, and estradiol (E2) in a cohort of late post-menopausal women. We examined 512 women ≥ 65 years from the InCHIANTI study. Retinol, α-caroten, β-caroten, β-criptoxantin, lutein, zeaxanthin, and lycopene levels were assayed at enrollment (1998-2000) by High-Performance Liquid Chromatography. Estradiol and testosterone (T) levels were assessed by Radioimmunometry (RIA) and testosterone-to-estradiol ratio (T/E2), as a proxy of aromatase activity, was also calculated. General linear models adjusted for age (Model 1) and further adjusted for other confounders including Body Mass Index (BMI) BMI, smoking, intake of energy, lipids, and vitamin A; C-Reactive Protein, insulin, total cholesterol, liver function, and testosterone (Model 2) were used to investigate the relationship between retinol, carotenoids, and E2 levels. To address the independent relationship between carotenoids and E2 levels, factors significantly associated with E2 in Model 2 were also included in a fully adjusted Model 3. After adjustment for age, α-carotene (β ± SE = -0.01 ± 0.004, p = 0.02) and β-carotene (β ± SE = -0.07 ± 0.02, p = 0.0007) were significantly and inversely associated with E2 levels. α-Carotene was also significantly and positively associated with T/E2 ratio (β ± SE = 0.07 ± 0.03, p = 0.01). After adjustment for other confounders (Model 2), the inverse relationship between α-carotene (β ± SE = -1.59 ± 0.61, p = 0.01), β-carotene (β ± SE = -0.29 ± 0.08, p = 0.0009), and E2 persisted whereas the

  10. Methoxychlor reduces estradiol levels by altering steroidogenesis and metabolism in mouse antral follicles in vitro

    International Nuclear Information System (INIS)

    Basavarajappa, Mallikarjuna S.; Craig, Zelieann R.; Hernandez-Ochoa, Isabel; Paulose, Tessie; Leslie, Traci C.; Flaws, Jodi A.

    2011-01-01

    The organochlorine pesticide methoxychlor (MXC) is a known endocrine disruptor that affects adult rodent females by causing reduced fertility, persistent estrus, and ovarian atrophy. Since MXC is also known to target antral follicles, the major producer of sex steroids in the ovary, the present study was designed to test the hypothesis that MXC decreases estradiol (E 2 ) levels by altering steroidogenic and metabolic enzymes in the antral follicles. To test this hypothesis, antral follicles were isolated from CD-1 mouse ovaries and cultured with either dimethylsulfoxide (DMSO) or MXC. Follicle growth was measured every 24 h for 96 h. In addition, sex steroid hormone levels were measured using enzyme-linked immunosorbent assays (ELISA) and mRNA expression levels of steroidogenic enzymes as well as the E 2 metabolic enzyme Cyp1b1 were measured using qPCR. The results indicate that MXC decreased E 2 , testosterone, androstenedione, and progesterone (P 4 ) levels compared to DMSO. In addition, MXC decreased expression of aromatase (Cyp19a1), 17β-hydroxysteroid dehydrogenase 1 (Hsd17b1), 17α-hydroxylase/17,20-lyase (Cyp17a1), 3β hydroxysteroid dehydrogenase 1 (Hsd3b1), cholesterol side-chain cleavage (Cyp11a1), steroid acute regulatory protein (Star), and increased expression of Cyp1b1 enzyme levels. Thus, these data suggest that MXC decreases steroidogenic enzyme levels, increases metabolic enzyme expression and this in turn leads to decreased sex steroid hormone levels. - Highlights: → MXC inhibits steroidogenesis → MXC inhibits steroidogenic enzymes → MXC induces metabolic enzymes

  11. High estradiol levels improve false memory rates and meta-memory in highly schizotypal women.

    Science.gov (United States)

    Hodgetts, Sophie; Hausmann, Markus; Weis, Susanne

    2015-10-30

    Overconfidence in false memories is often found in patients with schizophrenia and healthy participants with high levels of schizotypy, indicating an impairment of meta-cognition within the memory domain. In general, cognitive control is suggested to be modulated by natural fluctuations in oestrogen. However, whether oestrogen exerts beneficial effects on meta-memory has not yet been investigated. The present study sought to provide evidence that high levels of schizotypy are associated with increased false memory rates and overconfidence in false memories, and that these processes may be modulated by natural differences in estradiol levels. Using the Deese-Roediger-McDermott paradigm, it was found that highly schizotypal participants with high estradiol produced significantly fewer false memories than those with low estradiol. No such difference was found within the low schizotypy participants. Highly schizotypal participants with high estradiol were also less confident in their false memories than those with low estradiol; low schizotypy participants with high estradiol were more confident. However, these differences only approached significance. These findings suggest that the beneficial effect of estradiol on memory and meta-memory observed in healthy participants is specific to highly schizotypal individuals and might be related to individual differences in baseline dopaminergic activity. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  12. The role of histamine in estradiol-induced conditioned consumption reductions.

    Science.gov (United States)

    Hintiryan, Houri; Hayes, Unja L; Chambers, Kathleen C

    2005-01-31

    Conditioned consumption reductions (CCRs) develop toward novel taste stimuli as a consequence of associating those tastes with certain physiological changes. Few studies have focused on the neurochemical basis of this learned behavior. The purpose of these experiments was to reexamine the role of histamine in CCRs elicited by estradiol. Previous studies have suggested that histamine mediates CCRs induced by radiation, centrifugal rotation, and estradiol. However, because the animals were trained in a drug state, but tested in a nondrug state, it is possible that state-dependent learning confounded the results of these studies. The following series of experiments was performed to test this possibility for estradiol-induced CCRs. Implementing our own methodologies in Experiment 1, we demonstrated that an estradiol-induced CCR was blocked by treatment with the histamine 1 receptor blocker, chlorpheniramine maleate, before sucrose consumption during acquisition. In Experiment 2, identical states were maintained during acquisition and extinction by administering chlorpheniramine prior to sucrose exposure during both phases. The results indicated that chlorpheniramine blocked the estradiol-induced CCR. However, circumventing state-dependency in Experiment 3 by administering chlorpheniramine following exposure to sucrose during acquisition augmented the estradiol CCR. Taken together, the results of these experiments suggest that the ability of chlorpheniramine to abolish estradiol-induced CCRs is not due to state-dependency or to the antihistaminergic properties of chlorpheniramine. It is proposed that the results of all of the experiments can be accounted for by the aversive properties of chlorpheniramine.

  13. Insulin priming effect on estradiol-induced breast cancer metabolism and growth.

    Science.gov (United States)

    Wairagu, Peninah M; Phan, Ai N H; Kim, Min-Kyu; Han, Jeongwoo; Kim, Hyun-Won; Choi, Jong-Whan; Kim, Ki Woo; Cha, Seung-Kuy; Park, Kwang Hwa; Jeong, Yangsik

    2015-01-01

    Diabetes is a risk factor for breast cancer development and is associated with poor prognosis for breast cancer patients. However, the molecular and biochemical mechanisms underlying the association between diabetes and breast cancer have not been fully elucidated. Here, we investigated estradiol response in MCF-7 breast cancer cells with or without chronic exposure to insulin. We found that insulin priming is necessary and specific for estradiol-induced cancer cell growth, and induces anaplerotic shunting of glucose into macromolecule biosynthesis in the estradiol treated cells. Treatment with ERK or Akt specific inhibitors, U0126 or LY294002, respectively, suppressed estradiol-induced growth. Interestingly, molecular analysis revealed that estradiol treatment markedly increases expression of cyclin A and B, and decreases p21 and p27 in the insulin-primed cells. In addition, estradiol treatment activated metabolic genes in pentose phosphate (PPP) and serine biosynthesis pathways in the insulin-primed cells while insulin priming decreased metabolic gene expression associated with glucose catabolism in the breast cancer cells. Finally, we found that anti-diabetic drug metformin and AMPK ligand AICAR, but not thiazolidinediones (TZDs), specifically suppress the estradiol-induced cellular growth in the insulin-primed cells. These findings suggest that estrogen receptor (ER) activation under chronic hyperinsulinemic condition increases breast cancer growth through the modulation of cell cycle and apoptotic factors and nutrient metabolism, and further provide a mechanistic evidence for the clinical benefit of metformin use for ER-positive breast cancer patients with diabetes.

  14. Effects of estradiol and FSH on leptin levels in women with suppressed pituitary

    Directory of Open Access Journals (Sweden)

    Geber Selmo

    2012-06-01

    Full Text Available Abstract Background Female fertility depends on adequate nutrition and energy reserves, suggesting a correlation between the metabolic reserve and reproductive capacity. Leptin regulates body weight and energy homeostasis. The aim of this study was to investigate whether estradiol or FSH alone has a direct effect on the production of leptin. Methods A total of 64 patients submitted to controlled ovarian hyperstimulation with recombinant FSH for assisted reproduction and 20 patients using estradiol valerate for endometrial preparation for oocyte donation treatment were included in the study. All patients used GnRH analogues before starting treatment to achieve pituitary suppression. Blood samples for hormonal measurements were collected before starting and after completing the respective treatments. Data were analyzed statistically by the chi-square test, Student’s t-test and Pearson’s correlation test. Results We observed an elevation of serum leptin levels secondary to the increase in estradiol, in the absence of influence of any other ovarian or pituitary hormone. The rising rate of leptin levels was higher in women treated with recombinant FSH, which also had higher levels of estradiol, than in those treated with estradiol valerate. Conclusions This study demonstrates a correlation between serum levels of estradiol and leptin, suggesting that estradiol is an important regulator of leptin production and that its effects can be amplified by its association with FSH.

  15. Effects of estradiol and FSH on leptin levels in women with suppressed pituitary.

    Science.gov (United States)

    Geber, Selmo; Brandão, Augusto H F; Sampaio, Marcos

    2012-06-15

    Female fertility depends on adequate nutrition and energy reserves, suggesting a correlation between the metabolic reserve and reproductive capacity. Leptin regulates body weight and energy homeostasis. The aim of this study was to investigate whether estradiol or FSH alone has a direct effect on the production of leptin. A total of 64 patients submitted to controlled ovarian hyperstimulation with recombinant FSH for assisted reproduction and 20 patients using estradiol valerate for endometrial preparation for oocyte donation treatment were included in the study. All patients used GnRH analogues before starting treatment to achieve pituitary suppression. Blood samples for hormonal measurements were collected before starting and after completing the respective treatments. Data were analyzed statistically by the chi-square test, Student's t-test and Pearson's correlation test. We observed an elevation of serum leptin levels secondary to the increase in estradiol, in the absence of influence of any other ovarian or pituitary hormone. The rising rate of leptin levels was higher in women treated with recombinant FSH, which also had higher levels of estradiol, than in those treated with estradiol valerate. This study demonstrates a correlation between serum levels of estradiol and leptin, suggesting that estradiol is an important regulator of leptin production and that its effects can be amplified by its association with FSH.

  16. Estradiol concentrations and working memory performance in women of reproductive age.

    Science.gov (United States)

    Hampson, Elizabeth; Morley, Erin E

    2013-12-01

    Estrogen has been proposed to exert a regulatory influence on the working memory system via actions in the female prefrontal cortex. Tests of this hypothesis have been limited almost exclusively to postmenopausal women and pharmacological interventions. We explored whether estradiol discernibly influences working memory within the natural range of variation in concentrations characteristic of the menstrual cycle. The performance of healthy women (n=39) not using hormonal contraceptives, and a control group of age- and education-matched men (n=31), was compared on a spatial working memory task. Cognitive testing was done blind to ovarian status. Women were retrospectively classified into low- or high-estradiol groups based on the results of radioimmunoassays of saliva collected immediately before and after the cognitive testing. Women with higher levels of circulating estradiol made significantly fewer errors on the working memory task than women tested under low estradiol. Pearson's correlations showed that the level of salivary estradiol but not progesterone was correlated inversely with the number of working memory errors produced. Women tested at high levels of circulating estradiol tended to be more accurate than men. Superior performance by the high estradiol group was seen on the working memory task but not on two control tasks, indicating selectivity of the effects. Consistent with previous studies of postmenopausal women, higher levels of circulating estradiol were associated with better working memory performance. These results add further support to the hypothesis that the working memory system is modulated by estradiol in women, and show that the effects can be observed under non-pharmacological conditions. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Proliferation and chondrogenic differentiation of CD105-positive enriched rat synovium-derived mesenchymal stem cells in three-dimensional porous scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Qi Jun; Chen Anmin; You Hongbo; Li Kunpeng; Zhang Di; Guo Fengjing, E-mail: fjguo@tjh.tjmu.edu.cn [Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030 (China)

    2011-02-15

    Stem cell-based tissue engineering has provided an alternative strategy to treat cartilage lesions, and synovium-derived mesenchymal stem cells (SMSCs) are considered as a promising cell source for cartilage repair. In this study, the SMSCs were isolated from rat synovium, and CD105-positive (CD105{sup +}) cells were enriched using magnetic activated cell sorting. Sorted cells were subsequently seeded onto the chitosan-alginate composite three-dimensional (3D) porous scaffolds and cultured in chondrogenic culture medium in the presence of TGF-{beta}{sub 3} and BMP-2 for 2 weeks in vitro. After 2 weeks in culture, scanning electron microscopy results showed that cells attached and proliferated well on scaffolds, and secreted extracellular matrix were also observed. From day 7 to day 14, the total DNA and glucosaminoglycan content of the cells cultured in scaffolds were found to have increased significantly, and cell cycle analyses revealed that the percentage of cells in the S and G2/M phases increased and the percentage of cells in the G0/G1 phase decreased. Compared with non-sorted cells, the sorted cells cultured in scaffolds underwent more chondrogenic differentiation, as evidenced by higher expression of type II collagen and Sox9 at the protein and mRNA levels. The results suggest that CD105{sup +} enriched SMSCs may be a potential cell source for cartilage tissue engineering, and the chitosan-alginate composite 3D porous scaffold could provide a favorable microenvironment for supporting proliferation and chondrogenic differentiation of cells.

  18. Human umbilical cord Wharton's jelly stem cells undergo enhanced chondrogenic differentiation when grown on nanofibrous scaffolds and in a sequential two-stage culture medium environment.

    Science.gov (United States)

    Fong, Chui-Yee; Subramanian, Arjunan; Gauthaman, Kalamegam; Venugopal, Jayarama; Biswas, Arijit; Ramakrishna, Seeram; Bongso, Ariff

    2012-03-01

    The current treatments used for osteoarthritis from cartilage damage have their disadvantages of donor site morbidity, complicated surgical interventions and risks of infection and graft rejection. Recent advances in tissue engineering have offered much promise in cartilage repair but the best cell source and in vitro system have not as yet been optimised. Human bone marrow mesenchymal stem cells (hBMSCs) have thus far been the cell of choice. However, we derived a unique stem cell from the human umbilical cord Wharton's jelly (hWJSC) that has properties superior to hBMSCs in terms of ready availability, prolonged stemness characteristics in vitro, high proliferation rates, wide multipotency, non-tumorigenicity and tolerance in allogeneic transplantation. We observed enhanced cell attachment, cell proliferation and chondrogenesis of hWJSCs over hBMSCs when grown on PCL/Collagen nanoscaffolds in the presence of a two-stage sequential complex/chondrogenic medium for 21 days. Improvement of these three parameters were confirmed via inverted optics, field emission scanning electron microscopy (FESEM), MTT assay, pellet diameters, Alcian blue histology and staining, glycosaminglycans (GAG) and hyaluronic acid production and expression of key chondrogenic genes (SOX9, Collagen type II, COMP, FMOD) using immunohistochemistry and real-time polymerase chain reaction (qRT-PCR). In separate experiments we demonstrated that the 16 ng/ml of basic fibroblast growth factor (bFGF) present in the complex medium may have contributed to driving chondrogenesis. We conclude that hWJSCs are an attractive stem cell source for inducing chondrogenesis in vitro when grown on nanoscaffolds and exposed sequentially first to complex medium and then followed by chondrogenic medium.

  19. Synthetic protease substrate n-benzoyl-L-argininyl-p-nitroanilide activates specific binding of [3H]estradiol to a protein in rat pancreas: relationship of structure to activity

    International Nuclear Information System (INIS)

    Grossman, A.

    1984-01-01

    N-benzoyl-L-argininyl-p-nitroanilide (BAN), a synthetic substrate for trypsin-like proteolytic enzymes, is a potent activator of [ 3 H]estradiol-binding to a protein present in rat pancreas. When partially purified, this protein is almost devoid of [ 3 H]estradiol-binding activity in the absence of an endogenous accessory factor. BAN can mimic the natural coligand in this steroid binding reaction. The effect of BAN is specific since a number of derivatives of this substance are inactive or may even inhibit steroid binding. It is unlikely that BAN exerts this stimulatory action indirectly, possibly by preventing proteolytic inactivation of the [ 3 H]estradiol-binding protein, since preincubation of the protein in the absence of BAN resulted neither in reduced rate, nor extent, of steroid binding following BAN addition. Also, a number of protease inhibitors had no effect on the binding reaction. Of those inhibitors tested, only antipain significantly enhanced binding of [ 3 H]estradiol, but only about 20 percent as effectively as BAN. 13 references, 1 figure, 2 tables

  20. Estrogen alleviates neuropathic pain induced after spinal cord injury by inhibiting microglia and astrocyte activation.

    Science.gov (United States)

    Lee, Jee Youn; Choi, Hae Young; Ju, Bong-Gun; Yune, Tae Young

    2018-04-16

    Neuropathic pain after spinal cord injury (SCI) is developed in about 80% of SCI patients and there is no efficient therapeutic drug to alleviate SCI-induced neuropathic pain. Here we examined the effect of estrogen on SCI-induced neuropathic pain at below-level and its effect on neuroinflammation as underlying mechanisms. Neuropathic pain is developed at late phase after SCI and a single dose of 17β-estradiol (100, 300 μg/kg) were administered to rats with neuropathic pain after SCI through intravenous injection. As results, both mechanical allodynia and thermal hyperalgesia were significantly reduced by 17β-estradiol compared to vehicle control. Both microglia and astrocyte activation in the lamina I and II of L4-5 dorsal horn was also inhibited by 17β-estradiol. In addition, the levels of p-p38MAPK and p-ERK known to be activated in microglia and p-JNK known to be activated in astrocyte were significantly decreased by 17β-estradiol. Furthermore, the mRNA expression of inflammatory mediators such as Il-1β, Il-6, iNos, and Cox-2 was more attenuated in 17β-estradiol-treated group than in vehicle-treated group. Particularly, we found that the analgesic effect by 17β-estradiol was mediated via estrogen receptors, which are expressed in dorsal horn neurons. These results suggest that 17β-estradiol may attenuate SCI-induced neuropathic pain by inhibiting microglia and astrocyte activation followed inflammation. Copyright © 2018 Elsevier B.V. All rights reserved.

  1. -5p and -3p strands of miR-145 and miR-140 during mesenchymal stem cell chondrogenic differentiation.

    Science.gov (United States)

    Kenyon, Jonathan D; Sergeeva, Olga; Somoza, Rodrigo A; Li, Ming; Caplan, Arnold I; Khalil, Ahmad M; Lee, Zhenghong

    2018-04-20

    The chondrogenic differentiation of mesenchymal stem cells (MSCs) is mediated by transcription factors and small non-coding RNAs such as micro-RNAs (miRNAs). Each miRNA is initially transcribed as a long transcript, which matures to produce -5p and -3p strands. It is widely believed that the mature and functional miRNA from any given pre-miRNA, usually the -5p strand, is functional, while the opposing -3p strand is degraded. However, recent cartilage literature started to show functional -3p stands for a few miRNAs. This study aimed at examining both -5p and -3p strands of two key miRNAs miR-140 and miR-145 that are known to be involved in the chondrogenic differentiation of MSCs. The level (copy number) of both -5p and -3p strands of miR-145 and miR-140 along the timeline of MSC chondrogenic differentiation was determined by PCR. The gene expression profiles of several genes related to MSC chondrogenesis were compared with these miRNA profiles along the same timeline. While miR-145-3p is declining in step with miR-145-5p in pellet cultures during the process, the -3p strand is only 1% - 2% of the total miR-145 products. In contrast, the mature -3p and -5p products of miR-140 are found to increase with near equal molar expression throughout chondrogenic differentiation. Numerous genes are expressed by cartilage progenitor cells during development. One such target gene, Sox9 is a regulatory target of the dominant miR-145-5p, consistent with the data. Further experimental validations are warranted to confirm that ACAN, FOXO1 and RUNX3 as direct targets of miR-145-5p in the context of MSC chondrogenesis. Similarly, TRSP1 and ACAN are worth further validation as direct targets of miR-145-3p. For miR-140, SOX4 shall be further validated as a direct target of miR-140-5p while KLF4, PTHLH, and WNT5A can be validated as direct targets of miR-140-3p.

  2. 17-β-Estradiol Upregulates the Stress Response in Candida albicans: Implications for Microbial Virulence

    OpenAIRE

    C. O’Connor; M. Essmann; B. Larsen

    1998-01-01

    Objective: The influence of 17-β-estradiol on the stress response of Candida albicans was studied.Methods: The survival of clinical isolates of C. albicans treated with 17-β-estradiol after heat and oxidative stress was measured by viable plate counts. Cellular proteins were analyzed via SDSPAGE.Results: The heat stress response induced by 17-β-estradiol in C. albicans grown at 25 ℃ protected the organisms against the lethal temperature of 48.5 ℃, as shown by viable plate counts. 17-β-estradi...

  3. 17-beta-estradiol upregulates the stress response in Candida albicans: implications for microbial virulence.

    OpenAIRE

    O'Connor, C; Essmann, M; Larsen, B

    1998-01-01

    OBJECTIVE: The influence of 17-beta-estradiol on the stress response of Candida albicans was studied. METHODS: The survival of clinical isolates of C. albicans treated with 17-beta-estradiol after heat and oxidative stress was measured by viable plate counts. Cellular proteins were analyzed via SDS-PAGE. RESULTS: The heat stress response induced by 17-beta-estradiol in C. albicans grown at 25 degrees C protected the organisms against the lethal temperature of 48.5 degrees C, as shown by viabl...

  4. Blocking p38 signalling inhibits chondrogenesis in vitro but not ankylosis in a model of ankylosing spondylitis in vivo.

    Science.gov (United States)

    Braem, Kirsten; Luyten, Frank P; Lories, Rik J U

    2012-05-01

    To investigate p38 mitogen activated protein kinase (MAPK) signalling in an in vitro model of bone morphogenetic protein (BMP) and transforming growth factor β (TGFβ)-induced chondrogenesis and in vivo, with specific attention to its potential role in ankylosing enthesitis. Human periosteum-derived cells (hPDCs) were cultured in pellets and stimulated with BMP2 or TGFβ1 in the presence or absence of a p38 inhibitor SB203580 or proinflammatory cytokines. Chondrogenic differentiation was evaluated using quantitative PCR. Male DBA/1 mice from different litters were caged together at the age of 8 weeks and treated with SB203580 in both a preventive and therapeutic strategy. The mice were evaluated for prospective signs of arthritis and the toe joints were analysed histologically to assess disease severity. p38 inhibition by SB203580 and proinflammatory cytokines downregulated chondrogenic markers in pellet cultures stimulated by BMP2 or TGFβ1. In contrast, the in vivo experiments resulted in an increased clinical incidence of arthritis and pathology severity score, reflecting progression towards ankylosis in mice given SB203580. Inhibition of p38 inhibited chondrogenic differentiation of progenitor cells, showing that not only the SMAD signalling pathways and also alternative activation of MAPKs including p38 contribute to chondrogenesis. Such an inhibitory effect is not found in an in vivo model of joint ankylosis and spondyloarthritis. Increased incidence and severity of disease in preventive experiments and shifts in disease stages in a therapeutic experimental set-up suggest that specific inhibition of p38 may have deleterious rather than beneficial effects.

  5. Hypothesis testing with computational modeling: linking aromatase inhibition with plasma vitellogenin dynamics in fathead minnows

    Science.gov (United States)

    Fadrozole inhibits aromatase (CYP19A), a key enzyme that converts testosterone to estradiol (E2). In fish, E2 concentrations control hepatic synthesis ofthe glycolipoprotein vitellogenin (VTG), an egg yolk precursor protein essential to oocyte development and larval survival. Whe...

  6. Inhibition of neointima formation by local delivery of estrogen receptor alpha and beta specific agonists

    NARCIS (Netherlands)

    Krom, Y.D.; Pires, N.M.M.; Jukema, J.W.; Vries, M.R. de; Frants, R.R.; Havekes, L.M.; Dijk, K.W. van; Quax, P.H.A.

    2007-01-01

    Objective: Neointima formation is the underlying mechanism of (in-stent) restenosis. 17β-Estradiol (E2) is known to inhibit injury-induced neointima formation and post-angioplasty restenosis. Estrogen receptor alpha (ERα) has been demonstrated to mediate E2 anti-restenotic properties. However, the

  7. Topical estradiol does not interfere with the expression of the metalloproteinase-1 enzyme in photo exposed skin cells Estradiol tópico não interfere na expressão da enzima metaloproteinase-1 em células da pele fotoexposta

    Directory of Open Access Journals (Sweden)

    Luciana Neder

    2012-02-01

    Full Text Available BACKGROUND: In postmenopausal women there is a rapid destruction of dermal collagen, resulting in accelerated skin ageing, which is manifested by cutaneous atrophy, increased number and depth of wrinkles and sagging. This accelerated catabolism of the collagen is due to estrogen deficiency and increased synthesis of the metalloproteinase-1 enzyme, which degrades the dermal collagen. OBJECTIVES: To assess whether the use of topical estradiol 0.05% cream on photo exposed skin can inhibit the expression of the metalloproteinase-1 enzyme on the dermis and subsequently the rapid loss of collagen in women after menopause. METHODS: We included 40 postmenopausal women without hormone replacement therapy. Information about lifestyle, lipid profile, blood glucose level, thyroid hormones, mammography, Pap smear and transvaginal ultrasound were obtained to rule out associated diseases. Skin biopsy of the right preauricular region was performed before and after treatment with topical estradiol 0.05% for 30 days. The biopsy specimens were subjected to immunohistochemistry to identify the expression of the metalloproteinase-1 enzyme. RESULTS: There was no statistically significant difference on the expression of the metalloproteinase-1 enzyme in keratinocytes, fibroblasts and endothelial cells before and after treatment with topical estradiol for 30 days. CONCLUSION: Treatment with estradiol 0.05% cream, in photo exposed skin for 30 days, does not inhibit the production of metalloproteinase-1.FUNDAMENTOS: Na pós-menopausa, ocorre rápida destruição do colágeno dérmico, com consequente envelhecimento acelerado da pele, que se expressa com atrofia cutânea, aumento do número e da profundidade das rugas e flacidez. Esse catabolismo acelerado do colágeno ocorre por deficiência estrogênica e aumento na síntese da enzima metaloproteinase-1, que degrada o colágeno dérmico. OBJETIVOS: Avaliar se o uso de estradiol tópico a 0,05% em creme na pele

  8. Estradiol to testosterone ratio in metabolic syndrome men aged started 40 years above

    Science.gov (United States)

    Kusuma, R.; Siregar, Y.; Mardianto

    2018-03-01

    Disruption of adipose tissue, an endocrine organ, could turn out into the so-called metabolic syndrome. Aging men with lowering testosterone were related to metabolic syndrome and excessive aromatase activity in adipose tissue would increase estradiol level. This study hypothesized that estradiol to testosterone ratio is increasedin aging, metabolic syndrome men. A total of 52 men were randomly recruited for this study. A blood samplewas drawn before 11.00 AM after 10 hoursof overnight fasting, then aliquot serum kept in -20°C pending the research. Subjects were divided evenly into the metabolic syndrome and nonmetabolicsyndrome group. The hormonal assaywas measured on the day of research. Then examined with student t-test. Estradiol level in metabolic syndrome group was increased, but insignificant differ to the other group. Testosterone level decreased and significantly different between groups. In conclusion, estradiol to testosterone ratio was increased in themetabolic syndrome group but insignificant.

  9. The pathway of estradiol-induced apoptosis in patients with systemic lupus erythematosus.

    Science.gov (United States)

    Rastin, Maryam; Hatef, Mohammad Reza; Tabasi, Nafisseh; Mahmoudi, Mahmoud

    2012-03-01

    Systemic lupus erythematosus (SLE) is a disease with unknown etiology. The pathologic role of sex hormones and apoptosis in SLE has often been discussed. We studied the effects of estradiol in the pathway of induced apoptosis in Iranian SLE patients. T lymphocytes from 35 SLE patients and 20 age-matched controls were isolated and cultured in the presence of 10(-8) M 17-β estradiol. The expression levels of Fas, Fas ligand (FasL), Bcl-2, caspase-8, and caspase-9 mRNAs were determined semiquantitatively in comparison to the expression level of beta actin RNA. Estradiol exposure did not have any significant effects on the expression levels of Fas, Bcl-2, and caspase-9 in SLE patients and controls. However, the expression levels of FasL and caspase-8 were significantly increased in SLE patients, but not in controls. This suggests the probable involvement of extrinsic apoptosis pathway in estradiol-induced apoptosis in SLE.

  10. Estradiol or fluoxetine alters depressive behavior and tryptophan hydroxylase in rat raphe.

    Science.gov (United States)

    Yang, Fu-Zhong; Wu, Yan; Zhang, Wei-Guo; Cai, Yi-Yun; Shi, Shen-Xun

    2010-03-10

    The effects of 17beta-estradiol and fluoxetine on behavior of ovariectomized rats subjected to the forced swimming test and the expression of tryptophan hydroxylase (TPH) in dorsal and median raphe were investigated, respectively through time sampling technique of behavior scoring and immunohistochemistry. Both estradiol and fluoxetine increased swimming and decreased immobility in the forced swimming test. The forced swimming stress decreased integrated optical density of TPH-positive regions in dorsal and median raphe. Both estradiol and fluoxetine administration prevented integrated optical density of TPH-positive regions from being decreased by forced swimming stress. These observations suggest that both estradiol and fluoxetine have protective bearing on ovariectomized rats enduring forced swimming stress.

  11. Effects of estradiol on worm burden and peripheral leukocytes in Parastrongylus malaysiensis-infected rats.

    Science.gov (United States)

    Kamis, A B; Ahmad, R A; Badrul-Munir, M Z

    1994-01-01

    Gonadectomized male laboratory rats were given 0.06 mg/kg estradiol benzoate daily for 14 days before being inoculated with 50 third-stage larvae of Parastrongylus malaysiensis. Hormone treatment was continued until the rats were killed. The numbers of larvae in the brain and of adult worms in the pulmonary area of the rats were determined every 7 days after the inoculation. It was found that the rats treated daily with estradiol benzoate had significantly and consistently higher numbers of larvae and adult worms as compared with the controls. The number of total leukocytes increased significantly after the rats were infected. The results show that estradiol-treated rats become susceptible to P. malaysiensis infection, which may indicate that the immunosuppressive effects of testosterone observed in earlier studies may partly be caused by estradiol that was peripherally aromatized from testosterone.

  12. Evaluation of the Discriminative Potential of a Novel Biomarker for Estradiol Treatments in Bovine Animals

    NARCIS (Netherlands)

    Regal, P.; Blokland, M.H.; Fente, C.A.; Sterk, S.S.; Cepeda, A.; Ginkel, van L.A.

    2015-01-01

    The endogenous occurrence of natural hormones obstructs the application of classical targeted methods as confirmatory options. In the case of estradiol, the ultimate confirmation of its exogenous administration relies on gas chromatography coupled to combustion/isotope ratio mass spectrometry

  13. Equol inhibits growth, induces atresia, and inhibits steroidogenesis of mouse antral follicles in vitro

    International Nuclear Information System (INIS)

    Mahalingam, Sharada; Gao, Liying; Gonnering, Marni; Helferich, William; Flaws, Jodi A.

    2016-01-01

    Equol is a non-steroidal estrogen metabolite produced by microbial conversion of daidzein, a major soy isoflavone, in the gut of some humans and many animal species. Isoflavones and their metabolites can affect endogenous estradiol production, action, and metabolism, potentially influencing ovarian follicle function. However, no studies have examined the effects of equol on intact ovarian antral follicles, which are responsible for sex steroid synthesis and further development into ovulatory follicles. Thus, the present study tested the hypothesis that equol inhibits antral follicle growth, increases follicle atresia, and inhibits steroidogenesis in the adult mouse ovary. To test this hypothesis, antral follicles isolated from adult CD-1 mice were cultured with vehicle control (dimethyl sulfoxide; DMSO) or equol (600 nM, 6 μM, 36 μM, and 100 μM) for 48 and 96 h. Every 24 h, follicle diameters were measured to monitor growth. At 48 and 96 h, the culture medium was subjected to measurement of hormone levels, and the cultured follicles were subjected to gene expression analysis. Additionally, follicles were histologically evaluated for signs of atresia after 96 h of culture. The results indicate that equol (100 μM) inhibited follicle growth, altered the mRNA levels of bcl2-associated X protein and B cell leukemia/lymphoma 2, and induced follicle atresia. Further, equol decreased the levels of estradiol, testosterone, androstenedione, and progesterone, and it decreased mRNA levels of cholesterol side-chain cleavage, steroid 17-α-hydroxalase, and aromatase. Collectively, these data indicate that equol inhibits growth, increases atresia, and inhibits steroidogenesis of cultured mouse antral follicles. - Highlights: • Equol exposure inhibits antral follicle growth. • Equol exposure increases follicle atresia. • Equol exposure inhibits sex steroid hormone levels. • Equol exposure inhibits mRNA levels of certain steroidogenic enzymes.

  14. Equol inhibits growth, induces atresia, and inhibits steroidogenesis of mouse antral follicles in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Mahalingam, Sharada, E-mail: mahalin2@illinois.edu [Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois, 2001 S. Lincoln Ave, Urbana, IL 61802 (United States); Gao, Liying, E-mail: lgao@uiuc.edu [Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois, 2001 S. Lincoln Ave, Urbana, IL 61802 (United States); Gonnering, Marni, E-mail: mgonne2@illinois.edu [Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois, 2001 S. Lincoln Ave, Urbana, IL 61802 (United States); Helferich, William, E-mail: helferic@illinois.edu [Department of Food Science and Human Nutrition, University of Illinois, 905 S. Goodwin, Urbana, IL 61801 (United States); Flaws, Jodi A., E-mail: jflaws@illinois.edu [Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois, 2001 S. Lincoln Ave, Urbana, IL 61802 (United States)

    2016-03-15

    Equol is a non-steroidal estrogen metabolite produced by microbial conversion of daidzein, a major soy isoflavone, in the gut of some humans and many animal species. Isoflavones and their metabolites can affect endogenous estradiol production, action, and metabolism, potentially influencing ovarian follicle function. However, no studies have examined the effects of equol on intact ovarian antral follicles, which are responsible for sex steroid synthesis and further development into ovulatory follicles. Thus, the present study tested the hypothesis that equol inhibits antral follicle growth, increases follicle atresia, and inhibits steroidogenesis in the adult mouse ovary. To test this hypothesis, antral follicles isolated from adult CD-1 mice were cultured with vehicle control (dimethyl sulfoxide; DMSO) or equol (600 nM, 6 μM, 36 μM, and 100 μM) for 48 and 96 h. Every 24 h, follicle diameters were measured to monitor growth. At 48 and 96 h, the culture medium was subjected to measurement of hormone levels, and the cultured follicles were subjected to gene expression analysis. Additionally, follicles were histologically evaluated for signs of atresia after 96 h of culture. The results indicate that equol (100 μM) inhibited follicle growth, altered the mRNA levels of bcl2-associated X protein and B cell leukemia/lymphoma 2, and induced follicle atresia. Further, equol decreased the levels of estradiol, testosterone, androstenedione, and progesterone, and it decreased mRNA levels of cholesterol side-chain cleavage, steroid 17-α-hydroxalase, and aromatase. Collectively, these data indicate that equol inhibits growth, increases atresia, and inhibits steroidogenesis of cultured mouse antral follicles. - Highlights: • Equol exposure inhibits antral follicle growth. • Equol exposure increases follicle atresia. • Equol exposure inhibits sex steroid hormone levels. • Equol exposure inhibits mRNA levels of certain steroidogenic enzymes.

  15. Oxidative stress parameters induced by exposure to either cadmium or 17β-estradiol on Mytilus galloprovincialis hemocytes. The role of signaling molecules

    International Nuclear Information System (INIS)

    Koutsogiannaki, Sophia; Franzellitti, Silvia; Fabbri, Elena; Kaloyianni, Martha

    2014-01-01

    Highlights: •Oxidative parameters in Mytilus galloprovincialis hemocytes were measured. •Comparison between cadmium and 17β-estradiol cytotoxicity is discussed. •NHE, PKC, PI3-K, NADPH oxidase, NO synthase, JNK involvement was observed. •Protective role of cAMP is suggested. •Signaling molecules studied could constitute novel biomarkers. -- Abstract: The aim of the present study was to determine and compare the possible effects of exposure to an estrogen, 17β-estradiol and to a metal, cadmium on oxidative parameters of Mytilus galloprovincialis hemocytes and to elucidate the signaling pathways that probably mediate the studied effects exerted by these two chemicals. In addition, it was of interest to investigate if the studied parameters could constitute biomarkers for aquatic pollution monitoring. Our results suggest that micromolar concentrations of either cadmium or 17β-estradiol affected the redox status of mussels by modulating oxidative parameters and antioxidant enzymes gene expression in mussel M. galloprovincialis hemocytes. In particular, our results showed that treatment of hemocytes with either 5 μM of cadmium chloride or with 25 nM of 17β-estradiol for 30 min caused significant increased ROS production; this led to oxidative damage exemplified by significant increased DNA damage, protein carbonylation and lipid peroxidation, as well as increased mRNA levels of the antioxidant enzymes catalase (CAT), superoxide dismoutase (SOD) and glutathione S-transferase (GST). Furthermore, our results suggest that either cadmium or 17β-estradiol signal is mediated either through one of the already known pathways initiated by photatidyl-inositol 3-kinase (PI3 K) and reaching Na + /H + exchanger (NHE) probably through protein kinase C (PKC) or a kinase-mediated signaling pathway that involves in most of the cases NHE, PKC, Ca 2+ -dependent PKC isoforms, PI3-K, NADPH oxidase, nitric oxide (NO) synthase, c-Jun N-terminal kinase (JNK) and cyclic adenosine

  16. Oxidative stress parameters induced by exposure to either cadmium or 17β-estradiol on Mytilus galloprovincialis hemocytes. The role of signaling molecules

    Energy Technology Data Exchange (ETDEWEB)

    Koutsogiannaki, Sophia [Laboratory of Animal Physiology, Zoology Department, School of Biology, Faculty of Science, Aristotle University of Thessaloniki, 54124 Thessaloniki (Greece); Franzellitti, Silvia [University of Bologna, Interdepartment Centre for Environmental Science Research, via S. Alberto 163, 48123 Ravenna (Italy); Fabbri, Elena [University of Bologna, Interdepartment Centre for Environmental Science Research, via S. Alberto 163, 48123 Ravenna (Italy); University of Bologna, Department of Biological, Geological, and Environmental Sciences, via Selmi 3, 40100 Bologna (Italy); Kaloyianni, Martha, E-mail: kaloyian@bio.auth.gr [Laboratory of Animal Physiology, Zoology Department, School of Biology, Faculty of Science, Aristotle University of Thessaloniki, 54124 Thessaloniki (Greece)

    2014-01-15

    Highlights: •Oxidative parameters in Mytilus galloprovincialis hemocytes were measured. •Comparison between cadmium and 17β-estradiol cytotoxicity is discussed. •NHE, PKC, PI3-K, NADPH oxidase, NO synthase, JNK involvement was observed. •Protective role of cAMP is suggested. •Signaling molecules studied could constitute novel biomarkers. -- Abstract: The aim of the present study was to determine and compare the possible effects of exposure to an estrogen, 17β-estradiol and to a metal, cadmium on oxidative parameters of Mytilus galloprovincialis hemocytes and to elucidate the signaling pathways that probably mediate the studied effects exerted by these two chemicals. In addition, it was of interest to investigate if the studied parameters could constitute biomarkers for aquatic pollution monitoring. Our results suggest that micromolar concentrations of either cadmium or 17β-estradiol affected the redox status of mussels by modulating oxidative parameters and antioxidant enzymes gene expression in mussel M. galloprovincialis hemocytes. In particular, our results showed that treatment of hemocytes with either 5 μM of cadmium chloride or with 25 nM of 17β-estradiol for 30 min caused significant increased ROS production; this led to oxidative damage exemplified by significant increased DNA damage, protein carbonylation and lipid peroxidation, as well as increased mRNA levels of the antioxidant enzymes catalase (CAT), superoxide dismoutase (SOD) and glutathione S-transferase (GST). Furthermore, our results suggest that either cadmium or 17β-estradiol signal is mediated either through one of the already known pathways initiated by photatidyl-inositol 3-kinase (PI3 K) and reaching Na{sup +}/H{sup +} exchanger (NHE) probably through protein kinase C (PKC) or a kinase-mediated signaling pathway that involves in most of the cases NHE, PKC, Ca{sup 2+}-dependent PKC isoforms, PI3-K, NADPH oxidase, nitric oxide (NO) synthase, c-Jun N-terminal kinase (JNK) and

  17. Effects of estradiol and FSH on maturation of the testis in the hypogonadal (hpg mouse

    Directory of Open Access Journals (Sweden)

    Mayhew Terry M

    2008-01-01

    Full Text Available Abstract Background The hypogonadal (hpg mouse is widely used as an animal model with which to investigate the endocrine regulation of spermatogenesis. Chronic treatment of these GnRH-deficient mice with estradiol is known to induce testicular maturation and restore qualitatively normal spermatogenesis. The aim of the current studies was to investigate whether these effects of estradiol are direct effects in the testis, or indirect actions via paradoxical stimulation of FSH secretion from the pituitary gland. Methods Initially, Western blot and immunohistochemistry were used to analyse tissues from hpg mice to identify potential sites of action of estradiol. In the main study, hpg mice were treated for 50 days with either an estradiol implant or daily injections of recombinant human FSH, or a combination of both, to determine whether estradiol would have an additive or synergistic effect with FSH on testis development, as assessed by histological analysis and stereological quantification of Leydig, Sertoli and germ cell proliferation. Results Western blot analysis revealed ERα immunoreactive bands of appropriate molecular weight in extracts of testis and pituitary glands from hpg mice, and immunohistochemical studies confirmed ERα in nuclei of anterior pituitary cells and Leydig and peritubular cells in hpg mice. Histological and morphometric analyses revealed that estradiol treatment alone was as effective as FSH in promoting Sertoli cell production and proliferation of the seminiferous epithelium, resulting in the production of elongating spermatids. Combined estradiol and FSH treatment did not produce a greater effect than either treatment alone, though an increased dose of FSH significantly increased seminiferous tubule volume and testis weight and increase Sertoli cell numbers further within the same time frame. In contrast, estradiol caused substantial increases in the wet weight of the seminal vesicles, whereas FSH was without effect on

  18. Estradiol influences the mechanical properties of human fetal osteoblasts through cytoskeletal changes

    International Nuclear Information System (INIS)

    Muthukumaran, Padmalosini; Lim, Chwee Teck; Lee, Taeyong

    2012-01-01

    Highlights: ► Estradiol induced stiffness changes of osteoblasts were quantified using AFM. ► Estradiol causes significant decrease in the stiffness of osteoblasts. ► Decreased stiffness was caused by decreased density of f-actin network. ► Stiffness changes were not associated with mineralized matrix of osteoblasts. ► Estradiol increases inherent alkaline phosphatase activity of osteoblasts. -- Abstract: Estrogen is known to have a direct effect on bone forming osteoblasts and bone resorbing osteoclasts. The cellular and molecular effects of estrogen on osteoblasts and osteoblasts-like cells have been extensively studied. However, the effect of estrogen on the mechanical property of osteoblasts has not been studied yet. It is important since mechanical property of the mechanosensory osteoblasts could be pivotal to its functionality in bone remodeling. This is the first study aimed to assess the direct effect of estradiol on the apparent elastic modulus (E ∗ ) and corresponding cytoskeletal changes of human fetal osteoblasts (hFOB 1.19). The cells were cultured in either medium alone or medium supplemented with β-estradiol and then subjected to Atomic Force Microscopy indentation (AFM) to determine E ∗ . The underlying changes in cytoskeleton were studied by staining the cells with TRITC-Phalloidin. Following estradiol treatment, the cells were also tested for proliferation, alkaline phosphatase activity and mineralization. With estradiol treatment, E ∗ of osteoblasts significantly decreased by 43–46%. The confocal images showed that the changes in f-actin network observed in estradiol treated cells can give rise to the changes in the stiffness of the cells. Estradiol also increases the inherent alkaline phosphatase activity of the cells. Estradiol induced stiffness changes of osteoblasts were not associated with changes in the synthesized mineralized matrix of the cells. Thus, a decrease in osteoblast stiffness with estrogen treatment was

  19. Estradiol influences the mechanical properties of human fetal osteoblasts through cytoskeletal changes

    Energy Technology Data Exchange (ETDEWEB)

    Muthukumaran, Padmalosini [Department of Bioengineering, National University of Singapore (Singapore); Lim, Chwee Teck [Department of Bioengineering, National University of Singapore (Singapore); Department of Mechanical Engineering, National University of Singapore (Singapore); Mechanobiology Institute, National University of Singapore (Singapore); Singapore-MIT Alliance for Research and Technology (SMART), National University of Singapore (Singapore); Lee, Taeyong, E-mail: bielt@nus.edu.sg [Department of Bioengineering, National University of Singapore (Singapore)

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer Estradiol induced stiffness changes of osteoblasts were quantified using AFM. Black-Right-Pointing-Pointer Estradiol causes significant decrease in the stiffness of osteoblasts. Black-Right-Pointing-Pointer Decreased stiffness was caused by decreased density of f-actin network. Black-Right-Pointing-Pointer Stiffness changes were not associated with mineralized matrix of osteoblasts. Black-Right-Pointing-Pointer Estradiol increases inherent alkaline phosphatase activity of osteoblasts. -- Abstract: Estrogen is known to have a direct effect on bone forming osteoblasts and bone resorbing osteoclasts. The cellular and molecular effects of estrogen on osteoblasts and osteoblasts-like cells have been extensively studied. However, the effect of estrogen on the mechanical property of osteoblasts has not been studied yet. It is important since mechanical property of the mechanosensory osteoblasts could be pivotal to its functionality in bone remodeling. This is the first study aimed to assess the direct effect of estradiol on the apparent elastic modulus (E{sup Asterisk-Operator }) and corresponding cytoskeletal changes of human fetal osteoblasts (hFOB 1.19). The cells were cultured in either medium alone or medium supplemented with {beta}-estradiol and then subjected to Atomic Force Microscopy indentation (AFM) to determine E{sup Asterisk-Operator }. The underlying changes in cytoskeleton were studied by staining the cells with TRITC-Phalloidin. Following estradiol treatment, the cells were also tested for proliferation, alkaline phosphatase activity and mineralization. With estradiol treatment, E{sup Asterisk-Operator} of osteoblasts significantly decreased by 43-46%. The confocal images showed that the changes in f-actin network observed in estradiol treated cells can give rise to the changes in the stiffness of the cells. Estradiol also increases the inherent alkaline phosphatase activity of the cells. Estradiol induced stiffness

  20. Amniotic Mesenchymal Stromal Cells Exhibit Preferential Osteogenic and Chondrogenic Differentiation and Enhanced Matrix Production Compared With Adipose Mesenchymal Stromal Cells.

    Science.gov (United States)

    Topoluk, Natasha; Hawkins, Richard; Tokish, John; Mercuri, Jeremy

    2017-09-01

    Therapeutic efficacy of various mesenchymal stromal cell (MSC) types for orthopaedic applications is currently being investigated. While the concept of MSC therapy is well grounded in the basic science of healing and regeneration, little is known about individual MSC populations in terms of their propensity to promote the repair and/or regeneration of specific musculoskeletal tissues. Two promising MSC sources, adipose and amnion, have each demonstrated differentiation and extracellular matrix (ECM) production in the setting of musculoskeletal tissue regeneration. However, no study to date has directly compared the differentiation potential of these 2 MSC populations. To compare the ability of human adipose- and amnion-derived MSCs to undergo osteogenic and chondrogenic differentiation. Controlled laboratory study. MSC populations from the human term amnion were quantified and characterized via cell counting, histologic assessment, and flow cytometry. Differentiation of these cells in comparison to commercially purchased human adipose-derived mesenchymal stromal cells (hADSCs) in the presence and absence of differentiation media was evaluated via reverse transcription polymerase chain reaction (PCR) for bone and cartilage gene transcript markers and histology/immunohistochemistry to examine ECM production. Analysis of variance and paired t tests were performed to compare results across all cell groups investigated. The authors confirmed that the human term amnion contains 2 primary cell types demonstrating MSC characteristics-(1) human amniotic epithelial cells (hAECs) and (2) human amniotic mesenchymal stromal cells (hAMSCs)-and each exhibited more than 90% staining for MSC surface markers (CD90, CD105, CD73). Average viable hAEC and hAMSC yields at harvest were 2.3 × 10 6 ± 3.7 × 10 5 and 1.6 × 10 6 ± 4.7 × 10 5 per milliliter of amnion, respectively. As well, hAECs and hAMSCs demonstrated significantly greater osteocalcin ( P = .025), aggrecan ( P

  1. 17β-estradiol enhances memory duration in the main olfactory bulb in CD-1 mice.

    Science.gov (United States)

    Dillon, T Samuel; Fox, Laura C; Han, Crystal; Linster, Christiane

    2013-12-01

    Rodents rely heavily on odor detection, discrimination, and memory to locate food, find mates, care for pups, and avoid predators. Estrogens have been shown to increase memory retention in rodents performing spatial memory and object placement tasks. Here we evaluate the extent to which 17β-estradiol modulates memory formation and duration in the olfactory system. Adult CD-1 mice were gonadectomized and given either systemic 17β-estradiol replacement, local 17β-estradiol in the main olfactory bulb, or no replacement. Before performing the behavioral task the mice were given saline or PHTPP (an estrogen receptor β [ER-β] antagonist) via bilateral infusion into the main olfactory bulb. As the beta-type estrogen receptor (ER-β) is more abundant than the alpha-type estrogen receptor in the murine main olfactory bulb, the current study focuses on 17β-estradiol and its interactions with ERβ. Habituation, a simple, nonassociative learning task in which an animal is exposed to the same odor over successive presentations, was used to evaluate the animals' ability to detect odors and form an olfactory memory. To evaluate memory duration, we added a final trial of intertrial interval time (30 or 60 min) in which we presented the habituated odor. Neither surgical nor drug manipulation affected the ability of mice to detect or habituate to an odor. After habituation, gonadectomized 17β-estradiol-treated mice retained memory of an odor for 30 min, whereas non-estradiol-treated, 17β-estradiol+ERβ antagonist (PHTPP), and untreated male mice did not remember an odor 30 min after habituation. The results show that both systemic and local bulbar infusions of 17β-estradiol enhance odor memory duration in mice.

  2. TRANSDERMAL PERMEABILITY OF ESTRADIOL THROUGH THE HUMAN SKIN OF DIFFERENT BODY REGIONS IN VITRO

    Institute of Scientific and Technical Information of China (English)

    CHENGuo-Shen; GONGSai-Jun; DUJie; MARun-Zhen; ZHOURong-Rong; LIULiang-Chu

    1989-01-01

    Transdermal permeability of estradiol was carried out by using Valia-Chien double compartment permeation cells for the following regions of intact skin and skin without stratum corncum: chest, abdomen, hip, upper arm, thigh and back. The estradiol permeation rates and accumulative amounts within 72h in vitro were examined by HPLC. The results showed that the permeation rates of intact skin from different regions of the body

  3. Estradiol and song affect female zebra finch behavior independent of dopamine in the striatum

    OpenAIRE

    Svec, Lace A.; Lookingland, Keith J.; Wade, Juli

    2009-01-01

    Female songbirds display preferences for certain song characteristics, but the neural and hormonal mechanisms mediating these preferences are not fully clear. The present study sought to further explore the role of estradiol, as well as assess potential roles of dopaminergic systems, on behavioral responses to song. Adult female zebra finches were treated with estradiol and exposed to tutored or untutored song or silence. Behavior was quantified and neurochemistry of the nucleus accumbens and...

  4. 17β-Estradiol administration promotes delayed cutaneous wound healing in 40-week ovariectomised female mice.

    Science.gov (United States)

    Mukai, Kanae; Nakajima, Yukari; Urai, Tamae; Komatsu, Emi; Nasruddin; Sugama, Junko; Nakatani, Toshio

    2016-10-01

    This study investigated the effect of 17β-estradiol on wound healing in 40-week ovariectomised female mice. Thirty-six-week-old female mice were divided into three groups: medication with 17β-estradiol after ovariectomy (OVX + 17β-estradiol), ovariectomy (OVX) and sham (SHAM). The mice received two full-thickness wounds, and the OVX + 17β-estradiol group was administered 17β-estradiol at 0·01 g/day until healing. In the OVX + 17β-estradiol group, the ratio of wound area was significantly smaller than those of the OVX and SHAM groups on days 1-3, 5, 6, 8-12 and 9-12, respectively, the numbers of neutrophils and macrophages were significantly smaller than those on days 3 and 7, the ratio of re-epithelialisation was significantly higher than those on days 3 and 11, the ratio of myofibroblasts was significantly higher than those on day 11 and smaller on day 14, and the ratio of collagen fibres was significantly larger than that of the OVX group on days 7-14. We found that 17β-estradiol administration promotes cutaneous wound healing in 40-week female mice by reducing wound area, shortening inflammatory response, and promoting re-epithelialisation, collagen deposition and wound contraction. Our results suggest that cutaneous wound healing that is delayed because of ageing is promoted by exogenous and continuous 17β-estradiol administration. © 2014 The Authors. International Wound Journal © 2014 Medicalhelplines.com Inc and John Wiley & Sons Ltd.

  5. Interactions between estradiol and haloperidol on perseveration and reversal learning in amphetamine-sensitized female rats.

    Science.gov (United States)

    Almey, Anne; Arena, Lauren; Oliel, Joshua; Shams, Waqqas M; Hafez, Nada; Mancinelli, Cynthia; Henning, Lukas; Tsanev, Aleks; Brake, Wayne G

    2017-03-01

    There are sex differences associated with schizophrenia, as women exhibit later onset of the disorder, less severe symptomatology, and better response to antipsychotic medications. Estrogens are thought to play a role in these sex differences; estrogens facilitate the effects of antipsychotic medications to reduce the positive symptoms of schizophrenia, but it remains unclear whether estrogens protect against the cognitive symptoms of this disorder. Amphetamine sensitization is used to model some symptoms of schizophrenia in rats, including cognitive deficits like excessive perseveration and slower reversal learning. In this experiment female rats were administered a sensitizing regimen of amphetamine to mimic these cognitive symptoms. They were ovariectomized and administered either low or high estradiol replacement as well as chronic administration of the antipsychotic haloperidol, and were assessed in tests of perseveration and reversal learning. Results of these experiments demonstrated that, in amphetamine-sensitized rats, estradiol alone does not affect perseveration or reversal learning. However, low estradiol facilitates a 0.25mg/day dose of haloperidol to reduce perseveration and improve reversal learning. Combined high estradiol and 0.25mg/day haloperidol has no effect on perseveration or reversal learning, but high estradiol facilitates the effects of 0.13mg/day haloperidol to reduce perseveration and improve reversal learning. Thus, in amphetamine-sensitized female rats, 0.25mg/day haloperidol only improved perseveration and reversal learning when estradiol was low, while 0.13mg/day haloperidol only improved these cognitive processes when estradiol was high. These findings suggest that estradiol facilitates the effects of haloperidol to improve perseveration and reversal learning in a dose-dependent manner. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Modulation of SHBG binding to testosterone and estradiol by sex and morbid obesity.

    Science.gov (United States)

    Grasa, María Del Mar; Gulfo, José; Camps, Núria; Alcalá, Rosa; Monserrat, Laura; Moreno-Navarrete, José María; Ortega, Francisco José; Esteve, Montserrat; Remesar, Xavier; Fernández-López, José Antonio; Fernández-Real, José Manuel; Alemany, Marià

    2017-04-01

    Sex hormone-binding globulin (SHBG) binds and transports testosterone and estradiol in plasma. The possibility that SHBG is a mixture of transporting proteins has been postulated. We analyzed in parallel the effects of obesity status on the levels and binding capacity of circulating SHBG and their relationship with testosterone and estradiol. Anthropometric measures and plasma were obtained from apparently healthy young (i.e. 35 ± 7 years) premenopausal women ( n =  32) and men ( n =  30), with normal weight and obesity (BMI >30 kg/m 2 ). SHBG protein (Western blot), as well as the plasma levels of testosterone, estradiol, cortisol and insulin (ELISA) were measured. Specific binding of estradiol and testosterone to plasma SHBG was analyzed using tritium-labeled hormones. Significant differences in SHBG were observed within the obesity status and gender, with discordant patterns of change in testosterone and estradiol. In men, testosterone occupied most of the binding sites. Estrogen binding was much lower in all subjects. Lower SHBG of morbidly obese (BMI >40 kg/m 2 ) subjects affected testosterone but not estradiol. The ratio of binding sites to SHBG protein levels was constant for testosterone, but not for estradiol. The influence of gender was maximal in morbid obesity, with men showing the highest binding / SHBG ratios. The results reported here are compatible with SHBG being a mixture of at least two functionally different hormone-binding globulins, being affected by obesity and gender and showing different structure, affinities for testosterone and estradiol and also different immunoreactivity. © 2017 European Society of Endocrinology.

  7. Delivery of the Sox9 gene promotes chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells in an in vitro model

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Z.H. [Department of Otolaryngology - Head and Neck Surgery, The Second Hospital, Xi' an Jiaotong University, Xi' an (China); Li, X.L. [Department of Dermatology, The Second Hospital, Xi' an Jiaotong University, Xi' an (China); He, X.J. [Department of Orthopedics, The Second Hospital, Xi' an Jiaotong University, Xi' an (China); Wu, B.J.; Xu, M. [Department of Otolaryngology - Head and Neck Surgery, The Second Hospital, Xi' an Jiaotong University, Xi' an (China); Chang, H.M. [Department of Otolaryngology - Head and Neck Surgery, Affiliated Hospital of Xi' an Medical University, Xi' an (China); Zhang, X.H. [Department of Otolaryngology - Head and Neck Surgery, The Second Hospital, Xi' an Jiaotong University, Xi' an (China); Xing, Z. [Department of Clinical Dentistry, Faculty of Dentistry, Center for Clinical Dental Research, University of Bergen, Bergen (Norway); Jing, X.H.; Kong, D.M.; Kou, X.H.; Yang, Y.Y. [Department of Otolaryngology - Head and Neck Surgery, The Second Hospital, Xi' an Jiaotong University, Xi' an (China)

    2014-03-18

    SRY-related high-mobility-group box 9 (Sox9) gene is a cartilage-specific transcription factor that plays essential roles in chondrocyte differentiation and cartilage formation. The aim of this study was to investigate the feasibility of genetic delivery of Sox9 to enhance chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells (hUC-MSCs). After they were isolated from human umbilical cord blood within 24 h after delivery of neonates, hUC-MSCs were untreated or transfected with a human Sox9-expressing plasmid or an empty vector. The cells were assessed for morphology and chondrogenic differentiation. The isolated cells with a fibroblast-like morphology in monolayer culture were positive for the MSC markers CD44, CD105, CD73, and CD90, but negative for the differentiation markers CD34, CD45, CD19, CD14, or major histocompatibility complex class II. Sox9 overexpression induced accumulation of sulfated proteoglycans, without altering the cellular morphology. Immunocytochemistry demonstrated that genetic delivery of Sox9 markedly enhanced the expression of aggrecan and type II collagen in hUC-MSCs compared with empty vector-transfected counterparts. Reverse transcription-polymerase chain reaction analysis further confirmed the elevation of aggrecan and type II collagen at the mRNA level in Sox9-transfected cells. Taken together, short-term Sox9 overexpression facilitates chondrogenesis of hUC-MSCs and may thus have potential implications in cartilage tissue engineering.

  8. Alternative splicing targeting the hTAF4-TAFH domain of TAF4 represses proliferation and accelerates chondrogenic differentiation of human mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Jekaterina Kazantseva

    Full Text Available Transcription factor IID (TFIID activity can be regulated by cellular signals to specifically alter transcription of particular subsets of genes. Alternative splicing of TFIID subunits is often the result of external stimulation of upstream signaling pathways. We studied tissue distribution and cellular expression of different splice variants of TFIID subunit TAF4 mRNA and biochemical properties of its isoforms in human mesenchymal stem cells (hMSCs to reveal the role of different isoforms of TAF4 in the regulation of proliferation and differentiation. Expression of TAF4 transcripts with exons VI or VII deleted, which results in a structurally modified hTAF4-TAFH domain, increases during early differentiation of hMSCs into osteoblasts, adipocytes and chondrocytes. Functional analysis data reveals that TAF4 isoforms with the deleted hTAF4-TAFH domain repress proliferation of hMSCs and preferentially promote chondrogenic differentiation at the expense of other developmental pathways. This study also provides initial data showing possible cross-talks between TAF4 and TP53 activity and switching between canonical and non-canonical WNT signaling in the processes of proliferation and differentiation of hMSCs. We propose that TAF4 isoforms generated by the alternative splicing participate in the conversion of the cellular transcriptional programs from the maintenance of stem cell state to differentiation, particularly differentiation along the chondrogenic pathway.

  9. Delivery of the Sox9 gene promotes chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells in an in vitro model

    Directory of Open Access Journals (Sweden)

    Z.H. Wang

    2014-04-01

    Full Text Available SRY-related high-mobility-group box 9 (Sox9 gene is a cartilage-specific transcription factor that plays essential roles in chondrocyte differentiation and cartilage formation. The aim of this study was to investigate the feasibility of genetic delivery of Sox9 to enhance chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells (hUC-MSCs. After they were isolated from human umbilical cord blood within 24 h after delivery of neonates, hUC-MSCs were untreated or transfected with a human Sox9-expressing plasmid or an empty vector. The cells were assessed for morphology and chondrogenic differentiation. The isolated cells with a fibroblast-like morphology in monolayer culture were positive for the MSC markers CD44, CD105, CD73, and CD90, but negative for the differentiation markers CD34, CD45, CD19, CD14, or major histocompatibility complex class II. Sox9 overexpression induced accumulation of sulfated proteoglycans, without altering the cellular morphology. Immunocytochemistry demonstrated that genetic delivery of Sox9 markedly enhanced the expression of aggrecan and type II collagen in hUC-MSCs compared with empty vector-transfected counterparts. Reverse transcription-polymerase chain reaction analysis further confirmed the elevation of aggrecan and type II collagen at the mRNA level in Sox9-transfected cells. Taken together, short-term Sox9 overexpression facilitates chondrogenesis of hUC-MSCs and may thus have potential implications in cartilage tissue engineering.

  10. Macroautophagy and Selective Mitophagy Ameliorate Chondrogenic Differentiation Potential in Adipose Stem Cells of Equine Metabolic Syndrome: New Findings in the Field of Progenitor Cells Differentiation.

    Science.gov (United States)

    Marycz, Krzysztof; Kornicka, Katarzyna; Grzesiak, Jakub; Śmieszek, Agnieszka; Szłapka, Jolanta

    2016-01-01

    Equine metabolic syndrome (EMS) is mainly characterized by insulin resistance, obesity, and local or systemic inflammation. That unfriendly environment of adipose tissue has huge impact on stem cells population (ASC) residing within. In the present study, using molecular biology techniques and multiple imaging techniques (SEM, FIB-SEM, and confocal microscopy), we evaluated the impact of EMS on ASC viability and chondrogenic differentiation. Moreover, we visualized the mitochondrial network and dynamics in ASC CTRL and ASC EMS during control and chondrogenic conditions. In control conditions, ASC EMS were characterized by increased mitochondrial fission in comparison to ASC CTRL . We found that extensive remodeling of mitochondrial network including fusion and fission occurs during early step of differentiation. Moreover, we observed mitochondria morphology deterioration in ASC EMS . These conditions seem to cause autophagic shift in ASC EMS , as we observed increased accumulation of LAMP2 and formation of multiple autophagosomes in those cells, some of which contained dysfunctional mitochondria. "Autophagic" switch may be a rescue mechanism allowing ASC EMS to clear impaired by ROS proteins and mitochondria. Moreover it provides a precursors-to-macromolecules synthesis, especially during chondrogenesis. Our data indicates that autophagy in ASC EMS would be crucial for the quality control mechanisms and maintenance of cellular homeostasis ASC EMS allowing them to be in "stemness" status.

  11. Hypoxia Is a Critical Parameter for Chondrogenic Differentiation of Human Umbilical Cord Blood Mesenchymal Stem Cells in Type I/III Collagen Sponges

    Directory of Open Access Journals (Sweden)

    Tangni Gómez-Leduc

    2017-09-01

    Full Text Available Umbilical cord blood (UCB is an attractive alternative to bone marrow for isolation of mesenchymal stem cells (MSCs to treat articular cartilage defects. Here, we set out to determine the growth factors (bone morphogenetic protein 2 (BMP-2 and transforming growth factor-β (TGF-β1 and oxygen tension effects during chondrogenesis of human UCB-MSCs for cartilage engineering. Chondrogenic differentiation was induced using 3D cultures in type I/III collagen sponges with chondrogenic factors in normoxia (21% O2 or hypoxia (<5% O2 for 7, 14 and 21 days. Our results show that UCB-MSCs can be committed to chondrogenesis in the presence of BMP-2+TGF-β1. Normoxia induced the highest levels of chondrocyte-specific markers. However, hypoxia exerted more benefit by decreasing collagen X and matrix metalloproteinase-13 (MMP13 expression, two chondrocyte hypertrophy markers. However, a better chondrogenesis was obtained by switching oxygen conditions, with seven days in normoxia followed by 14 days in hypoxia, since these conditions avoid hypertrophy of hUCB-MSC-derived chondrocytes while maintaining the expression of chondrocyte-specific markers observed in normoxia. Our study demonstrates that oxygen tension is a key factor for chondrogenesis and suggests that UBC-MSCs 3D-culture should begin in normoxia to obtain a more efficient chondrocyte differentiation before placing them in hypoxia for chondrocyte phenotype stabilization. UCB-MSCs are therefore a reliable source for cartilage engineering.

  12. New Insights into Osteogenic and Chondrogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells and Their Potential Clinical Applications for Bone Regeneration in Pediatric Orthopaedics

    Directory of Open Access Journals (Sweden)

    Nicola Giuliani

    2013-01-01

    Full Text Available Human mesenchymal stem cells (hMSCs are pluripotent adult stem cells capable of being differentiated into osteoblasts, adipocytes, and chondrocytes. The osteogenic differentiation of hMSCs is regulated either by systemic hormones or by local growth factors able to induce specific intracellular signal pathways that modify the expression and activity of several transcription factors. Runt-related transcription factor 2 (Runx2 and Wnt signaling-related molecules are the major factors critically involved in the osteogenic differentiation process by hMSCs, and SRY-related high-mobility-group (HMG box transcription factor 9 (SOX9 is involved in the chondrogenic one. hMSCs have generated a great interest in the field of regenerative medicine, particularly in bone regeneration. In this paper, we focused our attention on the molecular mechanisms involved in osteogenic and chondrogenic differentiation of hMSC, and the potential clinical use of hMSCs in osteoarticular pediatric disease characterized by fracture nonunion and pseudarthrosis.

  13. Delivery of the Sox9 gene promotes chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells in an in vitro model

    International Nuclear Information System (INIS)

    Wang, Z.H.; Li, X.L.; He, X.J.; Wu, B.J.; Xu, M.; Chang, H.M.; Zhang, X.H.; Xing, Z.; Jing, X.H.; Kong, D.M.; Kou, X.H.; Yang, Y.Y.

    2014-01-01

    SRY-related high-mobility-group box 9 (Sox9) gene is a cartilage-specific transcription factor that plays essential roles in chondrocyte differentiation and cartilage formation. The aim of this study was to investigate the feasibility of genetic delivery of Sox9 to enhance chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells (hUC-MSCs). After they were isolated from human umbilical cord blood within 24 h after delivery of neonates, hUC-MSCs were untreated or transfected with a human Sox9-expressing plasmid or an empty vector. The cells were assessed for morphology and chondrogenic differentiation. The isolated cells with a fibroblast-like morphology in monolayer culture were positive for the MSC markers CD44, CD105, CD73, and CD90, but negative for the differentiation markers CD34, CD45, CD19, CD14, or major histocompatibility complex class II. Sox9 overexpression induced accumulation of sulfated proteoglycans, without altering the cellular morphology. Immunocytochemistry demonstrated that genetic delivery of Sox9 markedly enhanced the expression of aggrecan and type II collagen in hUC-MSCs compared with empty vector-transfected counterparts. Reverse transcription-polymerase chain reaction analysis further confirmed the elevation of aggrecan and type II collagen at the mRNA level in Sox9-transfected cells. Taken together, short-term Sox9 overexpression facilitates chondrogenesis of hUC-MSCs and may thus have potential implications in cartilage tissue engineering

  14. Different culture media affect growth characteristics, surface marker distribution and chondrogenic differentiation of human bone marrow-derived mesenchymal stromal cells.

    Science.gov (United States)

    Hagmann, Sebastien; Moradi, Babak; Frank, Sebastian; Dreher, Thomas; Kämmerer, Peer Wolfgang; Richter, Wiltrud; Gotterbarm, Tobias

    2013-07-30

    Bone marrow-derived mesenchymal stromal cells (BM-MSCs) play an important role in modern tissue engineering, while distinct variations of culture media compositions and supplements have been reported. Because MSCs are heterogeneous regarding their regenerative potential and their surface markers, these parameters were compared in four widely used culture media compositions. MSCs were isolated from bone marrow and expanded in four established cell culture media. MSC yield/1000 MNCs, passage time and growth index were observed. In P4, typical MSC surface markers were analysed by fluorescence cytometry. Additionally, chondrogenic, adipogenic and osteogenic differentiation potential were evaluated. Growth index and P0 cell yield varied importantly between the media. The different expansion media had a significant influence on the expression of CD10, CD90, CD105, CD140b CD146 and STRO-1. While no significant differences were observed regarding osteogenic and adipogenic differentiation, chondrogenic differentiation was superior in medium A as reflected by GAG/DNA content. The choice of expansion medium can have a significant influence on growth, differentiation potential and surface marker expression of mesenchymal stromal cells, which is of fundamental importance for tissue engineering procedures.

  15. Glycosaminoglycan-Mimetic Signals Direct the Osteo/Chondrogenic Differentiation of Mesenchymal Stem Cells in a Three-Dimensional Peptide Nanofiber Extracellular Matrix Mimetic Environment.

    Science.gov (United States)

    Arslan, Elif; Guler, Mustafa O; Tekinay, Ayse B

    2016-04-11

    Recent efforts in bioactive scaffold development focus strongly on the elucidation of complex cellular responses through the use of synthetic systems. Designing synthetic extracellular matrix (ECM) materials must be based on understanding of cellular behaviors upon interaction with natural and artificial scaffolds. Hence, due to their ability to mimic both the biochemical and mechanical properties of the native tissue environment, supramolecular assemblies of bioactive peptide nanostructures are especially promising for development of bioactive ECM-mimetic scaffolds. In this study, we used glycosaminoglycan (GAG) mimetic peptide nanofiber gel as a three-dimensional (3D) platform to investigate how cell lineage commitment is altered by external factors. We observed that amount of fetal bovine serum (FBS) presented in the cell media had synergistic effects on the ability of GAG-mimetic nanofiber gel to mediate the differentiation of mesenchymal stem cells into osteogenic and chondrogenic lineages. In particular, lower FBS concentration in the culture medium was observed to enhance osteogenic differentiation while higher amount FBS promotes chondrogenic differentiation in tandem with the effects of the GAG-mimetic 3D peptide nanofiber network, even in the absence of externally administered growth factors. We therefore demonstrate that mesenchymal stem cell differentiation can be specifically controlled by the combined influence of growth medium components and a 3D peptide nanofiber environment.

  16. Macroautophagy and Selective Mitophagy Ameliorate Chondrogenic Differentiation Potential in Adipose Stem Cells of Equine Metabolic Syndrome: New Findings in the Field of Progenitor Cells Differentiation

    Directory of Open Access Journals (Sweden)

    Krzysztof Marycz

    2016-01-01

    Full Text Available Equine metabolic syndrome (EMS is mainly characterized by insulin resistance, obesity, and local or systemic inflammation. That unfriendly environment of adipose tissue has huge impact on stem cells population (ASC residing within. In the present study, using molecular biology techniques and multiple imaging techniques (SEM, FIB-SEM, and confocal microscopy, we evaluated the impact of EMS on ASC viability and chondrogenic differentiation. Moreover, we visualized the mitochondrial network and dynamics in ASCCTRL and ASCEMS during control and chondrogenic conditions. In control conditions, ASCEMS were characterized by increased mitochondrial fission in comparison to ASCCTRL. We found that extensive remodeling of mitochondrial network including fusion and fission occurs during early step of differentiation. Moreover, we observed mitochondria morphology deterioration in ASCEMS. These conditions seem to cause autophagic shift in ASCEMS, as we observed increased accumulation of LAMP2 and formation of multiple autophagosomes in those cells, some of which contained dysfunctional mitochondria. “Autophagic” switch may be a rescue mechanism allowing ASCEMS to clear impaired by ROS proteins and mitochondria. Moreover it provides a precursors-to-macromolecules synthesis, especially during chondrogenesis. Our data indicates that autophagy in ASCEMS would be crucial for the quality control mechanisms and maintenance of cellular homeostasis ASCEMS allowing them to be in “stemness” status.

  17. The lowest-dose, extended-cycle combined oral contraceptive pill with continuous ethinyl estradiol in the United States: a review of the literature on ethinyl estradiol 20 µg/levonorgestrel 100 µg + ethinyl estradiol 10 µg

    Directory of Open Access Journals (Sweden)

    Sheila Krishnan

    2010-08-01

    Full Text Available Sheila Krishnan, Jessica KileyDepartment of Obstetrics and Gynecology, Northwestern University, Chicago, Illinois, USAAbstract: Extended-cycle oral contraceptives (OCs are increasing in popularity in the United States. A new extended-cycle OC that contains the lowest doses of ethinyl estradiol (EE and levonorgestrel (LNG + continuous EE throughout the cycle is now available. It provides 84 days of a low-dose, combined active pill containing levonorgestrel 100 µg and ethinyl estradiol 20 µg. Instead of 7 days of placebo following the active pills, the regimen delivers 7 days of ethinyl estradiol 10 µg. Existing studies reveal a similar efficacy and adverse effect profile compared with other extended-regimen OCs. Specifically, the unscheduled bleeding profile is similar to other extended-cycle OCs and improves with the increase in the duration of use. Although lower daily doses of hormonal exposure have potential benefit, to our knowledge, there are no published studies indicating that this specific regimen offers a lower incidence of hormone-related side effects or adverse events. In summary, this new extended-cycle OC provides patients a low-dose, extended-regimen OC option without sacrificing efficacy or tolerability.Keywords: continuous regimen, ethinyl estradiol, extended cycle, oral contraceptive

  18. Pituitary, ovarian and additional contraceptive effects of an estradiol-based combined oral contraceptive: results of a randomized, open-label study.

    Science.gov (United States)

    Endrikat, Jan; Parke, Susanne; Trummer, Dietmar; Serrani, Marco; Duijkers, Ingrid; Klipping, Christine

    2013-02-01

    The estrogen step-down/progestogen step-up 28-day estradiol valerate/dienogest (E(2)V/DNG) oral contraceptive effectively inhibits ovulation; however, limited data are available regarding its effects on estradiol (E2), progesterone, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) or its additional extraovarian contraceptive effects. In this secondary analysis, 100 women received E(2)V 3 mg on days 1-2, E(2)V 2 mg/DNG 2 mg on days 3-7, E(2)V 2 mg/DNG 3 mg on days 8-24, E(2)V 1 mg on days 25-26 and placebo on days 27-28 for one treatment cycle. Measures included the presence/absence of cervical mucus; endometrial thickness; and serum E2, progesterone, and gonadotropin levels. E2, progesterone, LH and FSH levels did not exhibit the typical ovulatory increase and remained relatively stable during the cycle. E(2)V/DNG reduced mean maximal endometrial thickness and proportion of women with visible cervical mucus. All parameters returned to pretreatment levels during the posttreatment cycle. E(2)V/DNG provides extraovarian contraceptive effects (reducing endometrial thickness and cervical mucus production) in addition to inhibiting ovulation, assuring contraceptive efficacy. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. Estradiol decreases iodide uptake by rat thyroid follicular FRTL-5 cells

    Directory of Open Access Journals (Sweden)

    Furlanetto T.W.

    2001-01-01

    Full Text Available Estradiol has well-known indirect effects on the thyroid. A direct effect of estradiol on thyroid follicular cells, increasing cell growth and reducing the expression of the sodium-iodide symporter gene, has been recently reported. The aim of the present investigation was to study the effect of estradiol on iodide uptake by thyroid follicular cells, using FRTL-5 cells as a model. Estradiol decreased basal iodide uptake by FRTL-5 cells from control levels of 2.490 ± 0.370 to 2.085 ± 0.364 pmol I-/µg DNA at 1 ng/ml (P<0.02, to 1.970 ± 0.302 pmol I-/µg DNA at 10 ng/ml (P<0.003, and to 2.038 ± 0.389 pmol I-/µg DNA at 100 ng/ml (P<0.02. In addition, 4 ng/ml estradiol decreased iodide uptake induced by 0.02 mIU/ml thyrotropin from 8.678 ± 0.408 to 7.312 ± 0.506 pmol I-/µg DNA (P<0.02. A decrease in iodide uptake by thyroid cells caused by estradiol has not been described previously and may have a role in goiter pathogenesis.

  20. The daidzein- and estradiol- induced anorectic action in CCK or leptin receptor deficiency rats.

    Science.gov (United States)

    Fujitani, Mina; Mizushige, Takafumi; Bhattarai, Keshab; Iwahara, Asami; Aida, Ryojiro; Kishida, Taro

    2015-01-01

    We investigated the effect of daidzein feeding and estradiol treatment on food intake in cholecystokinin-1 receptor (CCK1R) deficiency, leptin receptor (ObRb) deficiency rats and their wild-type rats. These rats underwent an ovariectomy or a sham operation. For the 5 week experiment, each rat was divided in three groups: control, daidzein (150 mg/kg diet), and estradiol (4.2 μg/rat/day) groups. In both CCK1R+ and CCK1R- rats, daidzein feeding and estradiol treatment significantly decreased food intake. Daidzein feeding significantly reduced food intake in ovariectomized ObRb- rats, although not in ObRb+ rats. Estradiol treatment significantly lowered food intake in ovariectomized ObRb+ and ObRb- rats. In the ovariectomized rats, estradiol treatment significantly increases uterine weight, while daidzein feeding did not change it, suggesting that daidzein might have no or weak estrogenic effect in our experiment. These results suggest that CCK1R and ObRb signalings were not essential for the daidzein- and estradiol-induced anorectic action.

  1. Sex, estradiol, and spatial memory in a food-caching corvid.

    Science.gov (United States)

    Rensel, Michelle A; Ellis, Jesse M S; Harvey, Brigit; Schlinger, Barney A

    2015-09-01

    Estrogens significantly impact spatial memory function in mammalian species. Songbirds express the estrogen synthetic enzyme aromatase at relatively high levels in the hippocampus and there is evidence from zebra finches that estrogens facilitate performance on spatial learning and/or memory tasks. It is unknown, however, whether estrogens influence hippocampal function in songbirds that naturally exhibit memory-intensive behaviors, such as cache recovery observed in many corvid species. To address this question, we examined the impact of estradiol on spatial memory in non-breeding Western scrub-jays, a species that routinely participates in food caching and retrieval in nature and in captivity. We also asked if there were sex differences in performance or responses to estradiol. Utilizing a combination of an aromatase inhibitor, fadrozole, with estradiol implants, we found that while overall cache recovery rates were unaffected by estradiol, several other indices of spatial memory, including searching efficiency and efficiency to retrieve the first item, were impaired in the presence of estradiol. In addition, males and females differed in some performance measures, although these differences appeared to be a consequence of the nature of the task as neither sex consistently out-performed the other. Overall, our data suggest that a sustained estradiol elevation in a food-caching bird impairs some, but not all, aspects of spatial memory on an innate behavioral task, at times in a sex-specific manner. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. mTOR is involved in 17β-estradiol-induced, cultured immature boar Sertoli cell proliferation via regulating the expression of SKP2, CCND1, and CCNE1.

    Science.gov (United States)

    Yang, Wei-Rong; Wang, Yong; Wang, Yi; Zhang, Jiao-Jiao; Zhang, Jia-Hua; Lu, Cheng; Wang, Xian-Zhong

    2015-04-01

    Mammalian target of rapamycin (mTOR) is known to be involved in mammalian cell proliferation, while S-phase kinase-associated protein 2 (SKP2) plays a vital role in the cell cycle. Within the testis, estrogen also plays an important role in Sertoli cell proliferation, although it is not clear how. The present study asked if mTOR is involved in 17β-estradiol-dependent Sertoli cell proliferation. We specifically assessed if extracellular signal-regulated kinase 1/2 (ERK1/2) and/or phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) exert convergent effects toward the activation of mTOR signaling, and if this signaling regulates the expression of SKP2 through retinoblastoma (RB) and early mitotic inhibitor 1 (EMI1) protein and on CCNE1 and CCND1 mRNA levels. Treatment with 17β-estradiol for 15-90 min activated mTOR, with mTOR phosphorylation peaking after 30 min. U0126 (5 μM), a specific inhibitor of (MEK1/2), and 10-DEBC (2 μM), a selective inhibitor of AKT, both significantly reduced 17β-estradiol-induced phosphorylation of mTOR. Rapamycin suppressed 17β-estradiol-induced Sertoli cell proliferation, appearing to act by reducing the abundance of SKP2, CCND1, and CCNE1 mRNA as well as RB and EMI1 protein. These data indicated that 17β-estradiol enhances Sertoli cell proliferation via mTOR activation, which involves both ERK1/2 and PI3K/AKT signaling. Activated mTOR subsequently increases SKP2 mRNA and protein expression by enhancing the expression of CCND1 and CCNE1, and inhibits SKP2 protein degradation by increasing EMI1 abundance. © 2015 Wiley Periodicals, Inc.

  3. The significance of estradiol metabolites in human corpus luteum physiology.

    Science.gov (United States)

    Devoto, Luigi; Henríquez, Soledad; Kohen, Paulina; Strauss, Jerome F

    2017-07-01

    The human corpus luteum (CL) is a temporary endocrine gland derived from the ovulated follicle. Its formation and limited lifespan is critical for steroid hormone production required to support menstrual cyclicity, endometrial receptivity for successful implantation, and the maintenance of early pregnancy. Endocrine and paracrine-autocrine molecular mechanisms associated with progesterone production throughout the luteal phase are critical for the development, maintenance, regression, and rescue by hCG which sustains CL function into early pregnancy. However, the signaling systems driving the regression of the primate corpus luteum in non-conception cycles are not well understood. Recently, there has been interest in the functional roles of estradiol metabolites (EMs), mostly in estrogen-producing tissues. The human CL produces a number of EMs, and it has been postulated that the EMs acting via paracrine-autocrine pathways affect angiogenesis or LH-mediated events. The present review describes advances in understanding the role of EMs in the functional lifespan and regression of the human CL in non-conception cycles. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Laccase mediated transformation of 17β-estradiol in soil

    International Nuclear Information System (INIS)

    Singh, Rashmi; Cabrera, Miguel L.; Radcliffe, David E.; Zhang, Hao; Huang, Qingguo

    2015-01-01

    It is known that 17β-estradiol (E2) can be transformed by reactions mediated by some oxidoreductases such as laccase in water. Whether or how such reactions can happen in soil is however unknown although they may significantly impact the environmental fate of E2 that is introduced to soil by land application of animal wastes. We herein studied the reaction of E2 in a model soil mediated by laccase, and found that the reaction behaviors differ significantly from those in water partly because of the dramatic difference in laccase stability. We also examined E2 transformation in soil using 14 C-labeling in combination with soil organic matter extraction and size exclusion chromatography, which indicated that applied 14 C radioactivity was preferably bound to humic acids. The study provides useful information for understanding the environmental fate of E2 and for developing a novel soil remediation strategy via enzyme-enhanced humification reactions. - Highlights: • E2 was effectively transformed in soil through reactions mediated by laccase. • The reaction behaviors in soil differ significantly from those in water. • E2 was preferably bound to the humic acids in soil. • Laccase treatment resulted in changes in the structures of the humic acids. - E2 was effectively transformed in soil by preferably binding to the humic acids through reactions mediated by laccase

  5. Estradiol protective role in atherogenesis through LDL structure modification

    International Nuclear Information System (INIS)

    Papi, Massimiliano; Ciasca, Gabriele; Maiorana, Alessandro; Maulucci, Giuseppe; Palmieri, Valentina; De Spirito, Marco; Brunelli, Roberto; Parasassi, Tiziana

    2016-01-01

    Relevant physiological functions are exerted by circulating low density lipoprotein (LDL) as well as eventual pathological processes triggering atherogenesis. Modulation of these functions can well be founded on modifications of LDL structure. Given its large dimension, multicomponent organization and strong interactions between the protein apoB-100 and lipids, determining LDL 3D structure remains a challenge. We propose a novel quantitative physical approach to this complex biological problem. We introduce a three-component model, fitted to small angle x-ray scattering data on LDL maintained in physiological conditions, able to achieve a consistent 3D structure. Unexpected features include three distinct protein domains protruding out of a sphere, quite rough in its surface, where several core lipid areas are exposed. All LDL components are affected by 17- β -estradiol (E2) binding to apoB-100. Mostly one of the three protruding protein domains, dramatically reducing its presence on the surface and with a consequent increase of core lipids’ exposure. This result suggests a structural basis for some E2 protecting roles and LDL physiological modifications. (paper)

  6. Kisspeptin system in ovariectomized mice: Estradiol and progesterone regulation.

    Science.gov (United States)

    Marraudino, Marilena; Martini, Mariangela; Trova, Sara; Farinetti, Alice; Ponti, Giovanna; Gotti, Stefano; Panzica, GianCarlo

    2018-06-01

    The kisspeptin system is clustered in two main groups of cell bodies (the periventricular region, RP3V and the arcuate nucleus, ARC) that send fibers mainly to the GnRH neurons and in a few other locations, including the paraventricular nucleus, PVN. In physiological conditions, gonadal hormones modulate the kisspeptin system with expression changes according to different phases of the estrous cycle: the highest being in estrus phase in RP3V and PVN (positive feedback), and in ARC during the diestrus phase (negative feedback). In this work we wanted to study these hormonal fluctuations during the estrous cycle, investigating the role played by progesterone (P) or estradiol (E 2 ), alone or together, on the kisspeptin system. Gonadectomized CD1 female mice were treated with P, E 2 or both (E 2  + P), following a timing of administration that emulates the different phases of estrous cycle, for two cycles of 4 days. As expected, the two cell groups were differentially affected by E 2 ; the RP3V group was positively influenced by E 2 (alone or with the P), whereas in the ARC the administration of E 2 did not affect the system. However P (alone) induced a rise in the kisspeptin immunoreactivity. All the treatments significantly affected the kisspeptin innervation of the PVN, with regional differences, suggesting that these fibers arrive from both RP3V and ARC nuclei. Copyright © 2018 Elsevier B.V. All rights reserved.

  7. Effect of oral contraceptives containing estradiol and nomegestrol acetate or ethinyl-estradiol and chlormadinone acetate on primary dysmenorrhea.

    Science.gov (United States)

    Grandi, Giovanni; Napolitano, Antonella; Xholli, Anjeza; Tirelli, Alessandra; Di Carlo, Costantino; Cagnacci, Angelo

    2015-10-01

    To study the three cycles effect on primary dysmenorrhea of the monophasic 24/4 estradiol/nomegestrol acetate (E2/NOMAC) and of the 21/7 ethinyl-estradiol/chlormadinone acetate (EE/CMA) oral contraceptive. The tolerability and the effect of both preparations on metabolism and health-related quality of life were also evaluated. Prospective observational cohort study. Tertiary gynecologic center for pelvic pain. Subjects with primary dysmenorrhea requiring an oral contraceptive, who spontaneously selected either E2/NOMAC (n = 20) or EE/CMA (n = 20). Visual Analogue Scale (VAS) score for dysmenorrhea, Short Form-36 questionnaire for health-related quality of life, lipoproteins and days of menstrual bleeding (withdrawal bleeding during oral contraceptive). Mean age and body mass index (BMI) were similar between the two groups. The final analysis was performed on 34 women, 15 in E2/NOMAC and 19 in EE/CMA group. Compliance with treatment was significantly higher with EE/CMA (100%) than E2/NOMAC (75%) (p = 0.02). Both treatments significantly (p dysmenorrhea, similarly (E2/NOMAC by a mean of 74.7%, EE/CMA by a mean of 78.4%; p = 0.973). Only E2/NOMAC significantly increased SF-36 score (p = 0.001), both in physical (p = 0.001) and mental domains (p = 0.004). The mean number of days of menstrual bleeding was significantly reduced in E2/NOMAC group (from 4.86 ± 1.20 d to 2.64 ± 1.59 d, p = 0.0005 versus baseline, p = 0.007 versus EE/CMA group). BMI did not vary in either group. E2/NOMAC did not change lipoproteins and apoproteins while EE/CMA increased total cholesterol (p = 0.0114), HDL-cholesterol (p = 0.0008), triglycerides (p = 0.002), apoprotein-A1 (Apo-A1; p = 0.0006) and apopoprotein-B (Apo-B; p = 0.008), decreasing LDL/HDL ratio (p = 0.024). Both oral contraceptives reduced similarly primary dysmenorrhea, with E2/NOMAC also reducing withdrawal bleedings and being neutral on lipid metabolism.

  8. Kisspeptin Expression in Guinea Pig Hypothalamus: Effects of 17β-Estradiol

    Science.gov (United States)

    Bosch, Martha A.; Xue, Changhui; Rønnekleiv, Oline K.

    2013-01-01

    Kisspeptin is essential for reproductive functions in humans. As a model for the human we have used the female guinea pig, which has a long ovulatory cycle similar to that of primates. Initially, we cloned a guinea pig kisspeptin cDNA sequence and subsequently explored the distribution and 17β-estradiol (E2) regulation of kisspeptin mRNA (Kiss1) and protein (kisspeptin) by using in situ hybridization, real-time PCR and immunocytochemistry. In ovariectomized females, Kiss1 neurons were scattered throughout the preoptic periventricular areas (PV), but the vast majority of Kiss1 neurons were localized in the arcuate nucleus (Arc). An E2 treatment that first inhibits (negative feedback) and then augments (positive feedback) serum luteinizing hormone (LH) increased Kiss1 mRNA density and number of cells expressing Kiss1 in the PV at both time points. Within the Arc, Kiss1 mRNA density was reduced at both time points. Quantitative real-time PCR confirmed the in situ hybridization results during positive feedback. E2 reduced the number of immunoreactive kisspeptin cells in the PV at both time points, perhaps an indication of increased release. Within the Arc, the kisspeptin immunoreactivity was decreased during negative feedback but increased during positive feedback. Therefore, it appears that in guinea pig both the PV and the Arc kisspeptin neurons act cooperatively to excite gonadotropin-releasing hormone (GnRH) neurons during positive feedback. We conclude that E2 regulation of negative and positive feedback may reflect a complex interaction of the kisspeptin circuitry, and both the PV and the Arc respond to hormone signals to encode excitation of GnRH neurons during the ovulatory cycle. PMID:22173890

  9. Catabolic factors and osteoarthritis-conditioned medium inhibit chondrogenesis of human mesenchymal stem cells.

    NARCIS (Netherlands)

    Heldens, G.T.H.; Blaney Davidson, E.N.; Vitters, E.L.; Schreurs, B.W.; Piek, E.; Berg, W.B. van den; Kraan, P.M. van der

    2012-01-01

    Articular cartilage has a very limited intrinsic repair capacity leading to progressive joint damage. Therapies involving tissue engineering depend on chondrogenic differentiation of progenitor cells. This chondrogenic differentiation will have to survive in a diseased joint. We postulate that

  10. A cyclized peptide derived from alpha fetoprotein inhibits the proliferation of ER-positive canine mammary cancer cells.

    Science.gov (United States)

    Torres, Cristian Gabriel; Pino, Ana María; Sierralta, Walter Daniel

    2009-06-01

    The effects of estradiol (E2) and of an AFP-derived cyclized peptide (cP) on the proliferation of primary cultures of cancer cells isolated from spontaneous canine mammary tumors were studied. The cellular response to E2 and cP was related to the expression of estradiol receptor (isoforms alpha and beta). In ER-positive cells, 2 nM estradiol increased cell proliferation and the phosphorylation of ERK1/2; 2 microg/ml cP inhibited all these effects. Estradiol also increased HER2 immunoreactivity in ER-positive cells, an effect that was reverted to its basal values by cP. Estradiol stimulated in these cells the release of MMP2 and MMP9 and the shedding of HB-EGF, effects that the cP did not affect. ER-negative cells were refractory to estradiol or cP. All canine mammary tumor cells in culture responded to treatments analogously to human mammary cancer cells. Our results support the proposal of cP as a new, potentially effective therapeutic agent for the management of mammary cancer.

  11. Impact of TGF-β family-related growth factors on chondrogenic differentiation of adipose-derived stem cells isolated from lipoaspirates and infrapatellar fat pads of osteoarthritic patients

    Directory of Open Access Journals (Sweden)

    E López-Ruiz

    2018-04-01

    Full Text Available The success of cell-based approaches for the treatment of cartilage defects requires an optimal autologous cell source with chondrogenic differentiation ability that maintains its differentiated properties and stability following implantation. The objective of this study was to compare the chondrogenic capacity of mesenchymal stem cells (MSCs isolated from lipoaspirates (ASCs and the infrapatellar fat pad (IFPSCs of osteoarthritic patients and treated with transforming growth factor (TGF-β family-related growth factors. Cells were cultured for 6 weeks in a 3D pellet culture system with the chimeric activin A/bone morphogenic protein (BMP-2 ligand (AB235, the chimeric nodal/BMP-2 ligand (NB260 or BMP-2. To investigate the stability of the new cartilage, ASCs-treated pellets were transplanted subcutaneously into severe combined immunodeficiency (SCID mice. Histological and immunohistochemical assessment confirmed that the growth factors induced cartilage differentiation in both isolated cell types. However, reverse transcription-quantitative PCR results showed that ASCs presented a higher chondrogenic potential than IFPSCs. In vivo results revealed that AB235-treated ASCs pellets were larger in size and could form stable cartilage-like tissue as compared to NB260-treated pellets, while BMP-2-treated pellets underwent calcification. The chondrogenic induction of ASCs by AB235 treatment was mediated by SMAD2/3 activation, as proved by immunofluorescence analysis. The results of this study indicated that the combination of ASCs and AB235 might lead to a cell-based cartilage regeneration treatment.

  12. Effects of chitosan and bioactive glass modifications of knitted and rolled polylactide-based 96/4 L/D scaffolds on chondrogenic differentiation of adipose stem cells.

    Science.gov (United States)

    Ahtiainen, Katja; Sippola, Laura; Nurminen, Manu; Mannerström, Bettina; Haimi, Suvi; Suuronen, Riitta; Hyttinen, Jari; Ylikomi, Timo; Kellomäki, Minna; Miettinen, Susanna

    2015-01-01

    The performance of biodegradable knitted and rolled 3-dimensional (3D) polylactide-based 96/4 scaffolds modified with bioactive glass (BaG) 13-93, chitosan and both was compared with regard to the viability, proliferation and chondrogenic differentiation of rabbit adipose stem cells (ASCs). Scaffold porosities were determined by micro-computed tomography (μCT). Water absorption and degradation of scaffolds were studied during 28-day hydrolysis in Tris-buffer. Viability, number and differentiation of ASCs in PLA96/4 scaffolds were examined in vitro. The dimensions of the scaffolds were maintained during hydrolysis and mass loss was detected only in the BaG13-93 containing scaffolds. ASCs adhered and proliferated on each scaffold type. Cell aggregation and expression of chondral matrix components improved in all scaffold types in chondrogenic medium. Signs of hypertrophy were detected in the modified scaffolds but not in the plain PLA96/4 scaffold. Chondrogenic differentiation was most enhanced in the presence of chitosan. These findings indicate that the plain P scaffold provided a good 3D-matrix for ASC proliferation whereas the addition of chitosan to the PLA96/4 scaffold induced chondrogenic differentiation independent of the medium. Accordingly, a PLA96/4 scaffold modified by chitosan could provide a functional and bioactive basis for tissue-engineered chondral implants. Copyright © 2012 John Wiley & Sons, Ltd.

  13. Comparison of effects of estradiol with those of octylmethoxycinnamate and 4-methylbenzylidene camphor on fat tissue, lipids and pituitary hormones

    International Nuclear Information System (INIS)

    Seidlova-Wuttke, Dana; Christoffel, Julie; Rimoldi, Guillermo; Jarry, Hubertus; Wuttke, Wolfgang

    2006-01-01

    Octylmethoxycinnamate (OMC) and 4-methylbenzylidene camphor (4MBC) are commercially used absorbers of ultraviolet (UV) light. In rats, they were shown to exert endocrine disrupting including uterotrophic, i.e. estrogenic effects. Estrogens have also metabolic effects, therefore the impact of oral application of the two UV absorbers at 2 doses for 3 months on lipids and hormones were compared with those of estradiol-17β (E2). E2, OMC and 4MBC reduced weight gain, the size of fat depots and serum leptin, a lipocyte-derived hormone, when compared to the ovariectomized control animals. Serum triglycerides were also reduced by the UV screens but not by E2. On the other hand, E2 and OMC reduced serum cholesterol, low density lipoproteins and high density lipoproteins; this effect was not shared by 4MBC. While E2 inhibited, OMC and 4MBC stimulated serum LH levels. In the uterus, both UV filters had mild stimulatory effects. 4MBC inhibited serum T4 resulting in increased serum TSH levels. It is concluded that OMC and 4MBC have effects on several metabolic parameters such as fat and lipid homeostasis as well as on thyroid hormone production. Many of these effects are not shared by E2. Hence, other than estrogen-receptive mechanisms may be responsible for these effects

  14. Estrogen inhibits RANKL-stimulated osteoclastic differentiation of human monocytes through estrogen and RANKL-regulated interaction of estrogen receptor-α with BCAR1 and Traf6

    International Nuclear Information System (INIS)

    Robinson, Lisa J.; Yaroslavskiy, Beatrice B.; Griswold, Reed D.; Zadorozny, Eva V.; Guo, Lida; Tourkova, Irina L.; Blair, Harry C.

    2009-01-01

    The effects of estrogen on osteoclast survival and differentiation were studied using CD14-selected mononuclear osteoclast precursors from peripheral blood. Estradiol at ∼ 1 nM reduced RANKL-dependent osteoclast differentiation by 40-50%. Osteoclast differentiation was suppressed 14 days after addition of RANKL even when estradiol was withdrawn after 18 h. In CD14+ cells apoptosis was rare and was not augmented by RANKL or by 17-β-estradiol. Estrogen receptor-α (ERα) expression was strongly down-regulated by RANKL, whether or not estradiol was present. Mature human osteoclasts thus cannot respond to estrogen via ERα. However, ERα was present in CD14+ osteoclast progenitors, and a scaffolding protein, BCAR1, which binds ERα in the presence of estrogen, was abundant. Immunoprecipitation showed rapid (∼ 5 min) estrogen-dependent formation of ERα-BCAR1 complexes, which were increased by RANKL co-treatment. The RANKL-signaling intermediate Traf6, which regulates NF-κB activity, precipitated with this complex. Reduction of NF-κB nuclear localization occurred within 30 min of RANKL stimulation, and estradiol inhibited the phosphorylation of IκB in response to RANKL. Inhibition by estradiol was abolished by siRNA knockdown of BCAR1. We conclude that estrogen directly, but only partially, curtails human osteoclast formation. This effect requires BCAR1 and involves a non-genomic interaction with ERα.

  15. Estrogen inhibits RANKL-stimulated osteoclastic differentiation of human monocytes through estrogen and RANKL-regulated interaction of estrogen receptor-{alpha} with BCAR1 and Traf6

    Energy Technology Data Exchange (ETDEWEB)

    Robinson, Lisa J., E-mail: robinsonlj@msx.upmc.edu [Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Yaroslavskiy, Beatrice B.; Griswold, Reed D.; Zadorozny, Eva V.; Guo, Lida; Tourkova, Irina L. [Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Blair, Harry C. [Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Veteran' s Affairs Medical Center, Pittsburgh, PA 15243 (United States)

    2009-04-15

    The effects of estrogen on osteoclast survival and differentiation were studied using CD14-selected mononuclear osteoclast precursors from peripheral blood. Estradiol at {approx} 1 nM reduced RANKL-dependent osteoclast differentiation by 40-50%. Osteoclast differentiation was suppressed 14 days after addition of RANKL even when estradiol was withdrawn after 18 h. In CD14+ cells apoptosis was rare and was not augmented by RANKL or by 17-{beta}-estradiol. Estrogen receptor-{alpha} (ER{alpha}) expression was strongly down-regulated by RANKL, whether or not estradiol was present. Mature human osteoclasts thus cannot respond to estrogen via ER{alpha}. However, ER{alpha} was present in CD14+ osteoclast progenitors, and a scaffolding protein, BCAR1, which binds ER{alpha} in the presence of estrogen, was abundant. Immunoprecipitation showed rapid ({approx} 5 min) estrogen-dependent formation of ER{alpha}-BCAR1 complexes, which were increased by RANKL co-treatment. The RANKL-signaling intermediate Traf6, which regulates NF-{kappa}B activity, precipitated with this complex. Reduction of NF-{kappa}B nuclear localization occurred within 30 min of RANKL stimulation, and estradiol inhibited the phosphorylation of I{kappa}B in response to RANKL. Inhibition by estradiol was abolished by siRNA knockdown of BCAR1. We conclude that estrogen directly, but only partially, curtails human osteoclast formation. This effect requires BCAR1 and involves a non-genomic interaction with ER{alpha}.

  16. Effect of Endogenous Androgens on 17β-Estradiol-Mediated Protection after Spinal Cord Injury in Male Rats

    OpenAIRE

    Kachadroka, Supatra; Hall, Alicia M.; Niedzielko, Tracy L.; Chongthammakun, Sukumal; Floyd, Candace L.

    2010-01-01

    Several groups have recently shown that 17β-estradiol is protective in spinal cord injury (SCI). Testosterone can be aromatized to 17β-estradiol and may increase estrogen-mediated protection. Alternatively, testosterone has been shown to increase excitotoxicity in models of central nervous system (CNS) injury. These experiments test the hypothesis that endogenous testosterone in male rats alters 17β-estradiol-mediated protection by evaluating a delayed administration over a clinically relevan...

  17. Behavioral effects of endogenous or exogenous estradiol and progesterone on cocaine sensitization in female rats

    Energy Technology Data Exchange (ETDEWEB)

    Souza, M.F. [Universidade Federal de Ciências da Saúde de Porto Alegre, Laboratório de Neurociência Comportamental, Porto Alegre, RS, Brasil, Laboratório de Neurociência Comportamental, Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre, RS (Brazil); Couto-Pereira, N.S. [Universidade Federal do Rio Grande do Sul, Instituto de Ciências Básicas da Saúde, Departamento de Bioquímica, Porto Alegre, RS, Brasil, Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS (Brazil); Freese, L.; Costa, P.A.; Caletti, G.; Bisognin, K.M. [Universidade Federal de Ciências da Saúde de Porto Alegre, Laboratório de Neurociência Comportamental, Porto Alegre, RS, Brasil, Laboratório de Neurociência Comportamental, Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre, RS (Brazil); Nin, M.S. [Universidade Federal de Ciências da Saúde de Porto Alegre, Laboratório de Neurociência Comportamental, Porto Alegre, RS, Brasil, Laboratório de Neurociência Comportamental, Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre, RS (Brazil); Instituto Porto Alegre, Centro Metodista do Sul, Curso de Farmácia, Porto Alegre, RS, Brasil, Curso de Farmácia, Centro Metodista do Sul, Instituto Porto Alegre, Porto Alegre, RS (Brazil); Gomez, R. [Universidade Federal do Rio Grande do Sul, Instituto de Ciências Básicas da Saúde, Departamento de Farmacologia, Porto Alegre, RS, Brasil, Departamento de Farmacologia, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS (Brazil); Barros, H.M.T. [Universidade Federal de Ciências da Saúde de Porto Alegre, Laboratório de Neurociência Comportamental, Porto Alegre, RS, Brasil, Laboratório de Neurociência Comportamental, Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre, RS (Brazil)

    2014-05-09

    Cocaine sensitization is a marker for some facets of addiction, is greater in female rats, and may be influenced by their sex hormones. We compared the modulatory effects of endogenous or exogenous estradiol and progesterone on cocaine-induced behavioral sensitization in 106 female rats. Ovariectomized female rats received progesterone (0.5 mg/mL), estradiol (0.05 mg/mL), progesterone plus estradiol, or the oil vehicle. Sham-operated control females received oil. Control and acute subgroups received injections of saline, while the repeated group received cocaine (15 mg/kg, ip) for 8 days. After 10 days, the acute and repeated groups received a challenge dose of cocaine, after which locomotion and stereotypy were monitored. The estrous cycle phase was evaluated and blood was collected to verify hormone levels. Repeated cocaine treatment induced overall behavioral sensitization in female rats, with increased locomotion and stereotypies. In detailed analysis, ovariectomized rats showed no locomotor sensitization; however, the sensitization of stereotypies was maintained. Only females with endogenous estradiol and progesterone demonstrated increased locomotor activity after cocaine challenge. Estradiol replacement enhanced stereotyped behaviors after repeated cocaine administration. Cocaine sensitization of stereotyped behaviors in female rats was reduced after progesterone replacement, either alone or concomitant with estradiol. The behavioral responses (locomotion and stereotypy) to cocaine were affected differently, depending on whether the female hormones were of an endogenous or exogenous origin. Therefore, hormonal cycling appears to be an important factor in the sensitization of females. Although estradiol increases the risk of cocaine sensitization, progesterone warrants further study as a pharmacological treatment in the prevention of psychostimulant abuse.

  18. Behavioral effects of endogenous or exogenous estradiol and progesterone on cocaine sensitization in female rats

    International Nuclear Information System (INIS)

    Souza, M.F.; Couto-Pereira, N.S.; Freese, L.; Costa, P.A.; Caletti, G.; Bisognin, K.M.; Nin, M.S.; Gomez, R.; Barros, H.M.T.

    2014-01-01

    Cocaine sensitization is a marker for some facets of addiction, is greater in female rats, and may be influenced by their sex hormones. We compared the modulatory effects of endogenous or exogenous estradiol and progesterone on cocaine-induced behavioral sensitization in 106 female rats. Ovariectomized female rats received progesterone (0.5 mg/mL), estradiol (0.05 mg/mL), progesterone plus estradiol, or the oil vehicle. Sham-operated control females received oil. Control and acute subgroups received injections of saline, while the repeated group received cocaine (15 mg/kg, ip) for 8 days. After 10 days, the acute and repeated groups received a challenge dose of cocaine, after which locomotion and stereotypy were monitored. The estrous cycle phase was evaluated and blood was collected to verify hormone levels. Repeated cocaine treatment induced overall behavioral sensitization in female rats, with increased locomotion and stereotypies. In detailed analysis, ovariectomized rats showed no locomotor sensitization; however, the sensitization of stereotypies was maintained. Only females with endogenous estradiol and progesterone demonstrated increased locomotor activity after cocaine challenge. Estradiol replacement enhanced stereotyped behaviors after repeated cocaine administration. Cocaine sensitization of stereotyped behaviors in female rats was reduced after progesterone replacement, either alone or concomitant with estradiol. The behavioral responses (locomotion and stereotypy) to cocaine were affected differently, depending on whether the female hormones were of an endogenous or exogenous origin. Therefore, hormonal cycling appears to be an important factor in the sensitization of females. Although estradiol increases the risk of cocaine sensitization, progesterone warrants further study as a pharmacological treatment in the prevention of psychostimulant abuse

  19. Behavioral effects of endogenous or exogenous estradiol and progesterone on cocaine sensitization in female rats

    Directory of Open Access Journals (Sweden)

    M.F. Souza

    2014-06-01

    Full Text Available Cocaine sensitization is a marker for some facets of addiction, is greater in female rats, and may be influenced by their sex hormones. We compared the modulatory effects of endogenous or exogenous estradiol and progesterone on cocaine-induced behavioral sensitization in 106 female rats. Ovariectomized female rats received progesterone (0.5 mg/mL, estradiol (0.05 mg/mL, progesterone plus estradiol, or the oil vehicle. Sham-operated control females received oil. Control and acute subgroups received injections of saline, while the repeated group received cocaine (15 mg/kg, ip for 8 days. After 10 days, the acute and repeated groups received a challenge dose of cocaine, after which locomotion and stereotypy were monitored. The estrous cycle phase was evaluated and blood was collected to verify hormone levels. Repeated cocaine treatment induced overall behavioral sensitization in female rats, with increased locomotion and stereotypies. In detailed analysis, ovariectomized rats showed no locomotor sensitization; however, the sensitization of stereotypies was maintained. Only females with endogenous estradiol and progesterone demonstrated increased locomotor activity after cocaine challenge. Estradiol replacement enhanced stereotyped behaviors after repeated cocaine administration. Cocaine sensitization of stereotyped behaviors in female rats was reduced after progesterone replacement, either alone or concomitant with estradiol. The behavioral responses (locomotion and stereotypy to cocaine were affected differently, depending on whether the female hormones were of an endogenous or exogenous origin. Therefore, hormonal cycling appears to be an important factor in the sensitization of females. Although estradiol increases the risk of cocaine sensitization, progesterone warrants further study as a pharmacological treatment in the prevention of psychostimulant abuse.

  20. Effects of chronic estradiol treatment on the thyroid gland structure and function of ovariectomized rats

    Directory of Open Access Journals (Sweden)

    Elgendy Mohamed S

    2009-08-01

    Full Text Available Abstract Background Estrogen therapy is widely used nowadays in women to treat many postmenopausal symptoms but it may have some undesirable effects due to multiple organs affection. So, the aim of this study was to determine the effects of chronic estradiol treatment on the structure and function of the thyroid gland in ovarictomized rats as a model simulating menopause. Findings Thirty adult female Wistar rats divided into three groups were used in this study; the first group was sham-operated, while the second and third groups were ovariectomized. The first and second groups were injected with olive oil while the third group was injected with estradiol dipropionate daily for three months, after that; hormonal assay for T3, T4, TSH and specimens of the thyroid were taken and processed to be examined by light and electron microscopy. The results of this study revealed that serum levels of T3 and T4 decreased in ovariectomized animals and significantly increased after estradiol treatment, while TSH increased in ovariectomized animals and decreased with estradiol treatment. Histological and morphometric study in ovariectomized group revealed marked accumulation of colloid in follicular lumens with decreased epithelial height in addition to increased connective tissue amount. After estradiol treatment the follicles became smaller in size, having small amount of colloid with increased epithelial height in addition to decreased connective tissue content. Ultrastructural study supported these results in addition to the presence of large amount of intracytoplasmic colloid vesicles after estradiol treatment. Conclusion Low estrogen level may lead to mild thyroidal hypofunction while estradiol treatment may lead to hyperactivity so it should be used very cautiously in the treatment of postmenopausal symptoms to avoid its undesirable stimulatory effect on the thyroid.

  1. Concentrations of 17beta-estradiol in Holstein whole milk.

    Science.gov (United States)

    Pape-Zambito, D A; Magliaro, A L; Kensinger, R S

    2007-07-01

    Some individuals have expressed concern about estrogens in food because of their potential to promote growth of estrogen-sensitive human cancer cells. Researchers have reported concentrations of estrogen in milk but few whole milk samples have been analyzed. Because estrogen associates with the fat phase of milk, the analysis of whole milk is an important consideration. The objectives of this study, therefore, were to quantify 17beta-estradiol (E2) in whole milk from dairy cows and to determine whether E2 concentrations in milk from cows in the second half of pregnancy were greater than that in milk from cows in the first half of pregnancy or in nonpregnant cows. Milk samples and weights were collected during a single morning milking from 206 Holstein cows. Triplicate samples were collected and 2 samples were analyzed for fat, protein, lactose, and somatic cell counts (SCC); 1 sample was homogenized and analyzed for E2. The homogenized whole milk (3 mL) was extracted twice with ethyl acetate and once with methanol. The extract was reconstituted in benzene:methanol (9:1, vol/vol) and run over a Sephadex LH-20 column to separate E2 from cholesterol and estrone before quantification using radioimmunoassay. Cows were classified as not pregnant (NP, n = 138), early pregnant (EP, 1 to 140 d pregnant, n = 47), or midpregnant (MP, 141 to 210 d pregnant, n = 21) at the time of milk sampling based on herd health records. Mean E2 concentration in whole milk was 1.4 +/- 0.2 pg/mL and ranged from nondetectable to 22.9 pg/mL. Milk E2 concentrations averaged 1.3, 0.9, and 3.0 pg/mL for NP, EP, and MP cows, respectively. Milk E2 concentrations for MP cows were greater and differed from those of NP and EP cows. Milk composition was normal for a Holstein herd in that log SCC values and percentages of fat, protein, and lactose averaged 4.9, 3.5, 3.1, and 4.8, respectively. Estradiol concentration was significantly correlated (r = 0.20) with percentage fat in milk. Mean milk yield was

  2. Differential expression of CCN-family members in primary human bone marrow-derived mesenchymal stem cells during osteogenic, chondrogenic and adipogenic differentiation

    Directory of Open Access Journals (Sweden)

    Hendrich Christian

    2005-03-01

    Full Text Available Abstract Background The human cysteine rich protein 61 (CYR61, CCN1 as well as the other members of the CCN family of genes play important roles in cellular processes such as proliferation, adhesion, migration and survival. These cellular events are of special importance within the complex cellular interactions ongoing in bone remodeling. Previously, we analyzed the role of CYR61/CCN1 as an extracellular signaling molecule in human osteoblasts. Since mesenchymal stem cells of bone marrow are important progenitors for various differentiation pathways in bone and possess increasing potential for regenerative medicine, here we aimed to analyze the expression of CCN family members in bone marrow-derived human mesenchymal stem cells and along the osteogenic, the adipogenic and the chondrogenic differentiation. Results Primary cultures of human mesenchymal stem cells were obtained from the femoral head of patients undergoing total hip arthroplasty. Differentiation into adipocytes and osteoblasts was done in monolayer culture, differentiation into chondrocytes was induced in high density cell pellet cultures. For either pathway, established differentiation markers and CCN-members were analyzed at the mRNA level by RT-PCR and the CYR61/CCN1 protein was analyzed by immunocytochemistry. RT-PCR and histochemical analysis revealed the appropriate phenotype of differentiated cells (Alizarin-red S, Oil Red O, Alcian blue, alkaline phosphatase; osteocalcin, collagen types I, II, IX, X, cbfa1, PPARγ, aggrecan. Mesenchymal stem cells expressed CYR61/CCN1, CTGF/CCN2, CTGF-L/WISP2/CCN5 and WISP3/CCN6. The CYR61/CCN1 expression decreased markedly during osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation. These results were confirmed by immuncytochemical analyses. WISP2/CCN5 RNA expression declined during adipogenic differentiation and WISP3/CCN6 RNA expression was markedly reduced in chondrogenic differentiation. Conclusion The

  3. Estradiol-induced gene expression in largemouth bass (Micropterus salmoides)

    Science.gov (United States)

    Bowman, C.J.; Kroll, K.J.; Gross, T.G.; Denslow, N.D.

    2002-01-01

    Vitellogenin (Vtg) and estrogen receptor (ER) gene expression levels were measured in largemouth bass to evaluate the activation of the ER-mediated pathway by estradiol (E2). Single injections of E2 ranging from 0.0005 to 5 mg/kg up-regulated plasma Vtg in a dose-dependent manner. Vtg and ER mRNAs were measured using partial cDNA sequences corresponding to the C-terminal domain for Vtg and the ligand-binding domain of ER?? sequences. After acute E2-exposures (2 mg/kg), Vtg and ER mRNAs and plasma Vtg levels peaked after 2 days. The rate of ER mRNA accumulation peaked 36-42 h earlier than Vtg mRNA. The expression window for ER defines the primary response to E2 in largemouth bass and that for Vtg a delayed primary response. The specific effect of E2 on other estrogen-regulated genes was tested during these same time windows using differential display RT-PCR. Specific up-regulated genes that are expressed in the same time window as Vtg were ERp72 (a membrane-bound disulfide isomerase) and a gene with homology to an expressed gene identified in zebrafish. Genes that were expressed in a pattern that mimics the ER include the gene for zona radiata protein ZP2, and a gene with homology to an expressed gene found in winter flounder. One gene for fibrinogen ?? was down-regulated and an unidentified gene was transiently up-regulated after 12 h of exposure and returned to basal levels by 48 h. Taken together these studies indicate that the acute molecular response to E2 involves a complex network of responses over time. ?? 2002 Elsevier Science Ireland Ltd. All rights reserved.

  4. Lipid profiling and transcriptomic analysis reveals a functional interplay between estradiol and growth hormone in liver.

    Directory of Open Access Journals (Sweden)

    Leandro Fernández-Pérez

    Full Text Available 17β-estradiol (E2 may interfere with endocrine, metabolic, and gender-differentiated functions in liver in both females and males. Indirect mechanisms play a crucial role because of the E2 influence on the pituitary GH secretion and the GHR-JAK2-STAT5 signaling pathway in the target tissues. E2, through its interaction with the estrogen receptor, exerts direct effects on liver. Hypothyroidism also affects endocrine and metabolic functions of the liver, rendering a metabolic phenotype with features that mimic deficiencies in E2 or GH. In this work, we combined the lipid and transcriptomic analysis to obtain comprehensive information on the molecular mechanisms of E2 effects, alone and in combination with GH, to regulate liver functions in males. We used the adult hypothyroid-orchidectomized rat model to minimize the influence of internal hormones on E2 treatment and to explore its role in male-differentiated functions. E2 influenced genes involved in metabolism of lipids and endo-xenobiotics, and the GH-regulated endocrine, metabolic, immune, and male-specific responses. E2 induced a female-pattern of gene expression and inhibited GH-regulated STAT5b targeted genes. E2 did not prevent the inhibitory effects of GH on urea and amino acid metabolism-related genes. The combination of E2 and GH decreased transcriptional immune responses. E2 decreased the hepatic content of saturated fatty acids and induced a transcriptional program that seems to be mediated by the activation of PPARα. In contrast, GH inhibited fatty acid oxidation. Both E2 and GH replacements reduced hepatic CHO levels and increased the formation of cholesterol esters and triacylglycerols. Notably, the hepatic lipid profiles were endowed with singular fingerprints that may be used to segregate the effects of different hormonal replacements. In summary, we provide in vivo evidence that E2 has a significant impact on lipid content and transcriptome in male liver and that E2 exerts a

  5. The anti-inflammatory effect of montelukast, a cysteinyl leukotriene receptor-1 antagonist, against estradiol-induced nonbacterial inflammation in the rat prostate.

    Science.gov (United States)

    Said, Mahmoud M; Bosland, Maarten C

    2017-02-01

    There is no standard treatment of chronic nonbacterial prostatitis in humans. The current study was aimed to investigate the effect of montelukast, an antagonist of cysteinyl leukotriene receptor-1, against estrogen-induced, nonbacterial lateral lobe-specific prostate inflammation in rats. Male Wistar rats were randomized into five groups of six rats, including sham controls (group 1) and castrated rats (group 2), whereas nonbacterial prostatitis (NBP) was induced in groups 3-5 by castration followed by a daily subcutaneous injection of estradiol (0.25 mg/kg body weight) for 30 days. The rats were left otherwise untreated (group 3) or received a daily oral administration of montelukast (1 and 10 mg/kg body weight for groups 4 and 5, respectively) from the 17th day after castration for two consecutive weeks. Compared with sham controls, induction of NBP led to a significant increase in serum leukotriene B 4 (LTB4), tumor necrosis factor alpha (TNF-α), and interleukin 6 (IL-6) levels, along with a significant upregulation in the transcript level of proinflammatory molecules (nuclear factor kappa beta [NF-κβ] and inducible nitric oxide synthase [iNOS]), chemokines (monocyte chemoattractant protein-1 [MCP-1] and eotaxin), and E-selectin in the lateral prostate. Histological examination revealed intense inflammation in the prostate with leukocyte infiltration and acinar degeneration following estradiol treatment of castrated rats. Montelukast significantly suppressed the increase in serum and prostate proinflammatory mediators/chemokines expression and abolished the histologically inflammatory changes in the lateral prostate. These findings indicate that montelukast inhibits estradiol-induced NBP in a rat model through anti-inflammatory mechanisms, suggesting its future beneficial effect for the treatment of clinical chronic NBP.

  6. 17β-estradiol exerts anticancer effects in anoikis-resistant hepatocellular carcinoma cell lines by targeting IL-6/STAT3 signaling

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Seulki, E-mail: sl10f@naver.com [Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul, 110-799 (Korea, Republic of); Lee, Minjong, E-mail: minjonglee2@naver.com [Division of Gastroenterology, Department of Internal Medicine, Kangwon National University Hospital, 156 Baengnyeong-ro, Chuncheon-si, Gangwon-do (Korea, Republic of); Kim, Jong Bin, E-mail: kkimjp@hanmail.net [Hormel Institute, University of Minnesota, 801 16th Ave NE, Austin, MN, 55912 (United States); Jo, Ara, E-mail: loveara0315@naver.com [Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul, 110-799 (Korea, Republic of); Cho, Eun Ju, E-mail: creatioex@gmail.com [Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul, 110-799 (Korea, Republic of); Yu, Su Jong, E-mail: ydoctor2@hanmail.net [Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul, 110-799 (Korea, Republic of); Lee, Jeong-Hoon, E-mail: pindra@empal.com [Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul, 110-799 (Korea, Republic of); Yoon, Jung-Hwan, E-mail: yoonjh@snu.ac.kr [Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul, 110-799 (Korea, Republic of); Kim, Yoon Jun, E-mail: yoonjun@snu.ac.kr [Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul, 110-799 (Korea, Republic of)

    2016-05-13

    17β-Estradiol (E2) has been proven to exert protective effects against HCC; however, its mechanism on HCC proliferation and suppression of invasion remains to be further explored. Because HCC up-regulates serum Interleukin-6 (IL-6) levels and Signal Transducer and Activator of Transcription 3 (STAT3), molecular agents that attenuate IL-6/STAT3 signaling can potentially suppress HCC development. In this study, we examined involvement of E2 in anoikis resistance that induces invasion capacities and chemo-resistance. Huh-BAT and HepG2 cells grown under anchorage-independent condition were selected. The anoikis-resistant (AR) cells showed stronger chemo-resistance against sorafenib, doxorubicin, 5-fluorouracil and cisplatin compared to adherent HCC cells. AR HCC cells exhibited decreased expression of E-cadherin and increased expression of the N-cadherin and vimentin compared to adherent HCC cells. We then demonstrated that E2 suppressed cell proliferation in AR HCC cells. IL-6 treatment enhanced invasive characteristics, and E2 reversed it. Regarding mechanism of E2, it decreased in the phosphorylation of STAT3 that overexpressed on AR HCC cells. The inhibitory effect of E2 on cell growth was accompanied with cell cycle arrest at G2/M phase and caspase-3/9/PARP activation through c-Jun N-terminal Kinase (JNK) phosphorylation. Taken together, these findings suggested that E2 inhibited the proliferation of AR HCC cells through down-regulation of IL-6/STAT3 signaling. Thus, E2 can be a potential therapeutic drug for treatment of metastatic or chemo-resistant HCC. -- Highlights: •Anoikis-resistant HCC cells characterized chemo-resistant and metastatic potentials. •17β-Estradiol down-regulated IL-6/STAT3 signaling in anoikis-resistant HCC cells. •17β-Estradiol suppressed cell proliferation by inducing G2/M phase arrest and apoptosis though JNK phosphorylation.

  7. Effects of estradiol on norepinephrine and prostaglandin efflux in medial basal hypothalamus of ovariectomized rats

    International Nuclear Information System (INIS)

    Cardinali, D.P.; Fernandez Pardal, J.; Gimeno, M.F.; Gimeno, A.L.

    1982-01-01

    The spontaneous and K + -stimulated efflux of norepinephrine (NE) and the release of PGE 2 and PGF 2 α were examined in medial basal hypothalamus (MBH) of ovariectomized rats killed before and during the LH release that follows estradiol treatment. As compared to vehicle-treated, ovariectomized rats, estradiol-primed rats exhibited a 60% more increase in K + -stimulated 3 H-overflow of MBH slices preloaded with 3 H-NE at morning hours (1000 hours). Estradiol treatment did not result in further increase of K + -induced 3 H release from MBH slices at the time of LH release (1700 hours), nor affected labelled NE release in occipital cortex slices. A significant difference between K + -stimulated NE release of vehicle-treated spayed rats killed at 1000 and 1700 hours was observed, the latter showing 54% more release upon stimulus. PGE 2 efflux was time-dependent being highest at the evening in both vehicle- and estradiol-treated animals. The MBH of estrogenized rats released significantly more PGE 2 at the evening as compared to the controls. The release of PGF 2 α remained essentially unchanged regardless of estradiol treatment or time of day. The present results offer additional support to the involvement of MBH catecholamines and prostaglandins in the mechanism of LH secretion in the rat. (author)

  8. Epoxiconazole-induced degeneration in rat placenta and the effects of estradiol supplementation.

    Science.gov (United States)

    Rey Moreno, Maria Cecilia; Fussell, Karma C; Gröters, Sibylle; Schneider, Steffen; Strauss, Volker; Stinchcombe, Stefan; Fegert, Ivana; Veras, Mariana; van Ravenzwaay, Bennard

    2013-06-01

    Epoxiconazole (CAS-No. 133855-98-8) was recently shown to cause both a marked depletion of maternal estradiol blood levels and a significantly increased incidence of late fetal mortality when administered to pregnant rats throughout gestation (GD 7-18 or 21); estradiol supplementation prevented this epoxiconazole effect in rats (Stinchcombe et al., 2013), indicating that epoxiconazole-mediated estradiol depletion is a critical key event for induction of late fetal resorptions in rats. For further elucidation of the mode of action, the placentas from these modified prenatal developmental toxicity experiments with 23 and 50 mg/kg bw/d epoxiconazole were subjected to a detailed histopathological examination. This revealed dose-dependent placental degeneration characterized by cystic dilation of maternal sinuses in the labyrinth, leading to rupture of the interhemal membrane. Concomitant degeneration occurred in the trophospongium. Both placentas supporting live fetuses and late fetal resorptions were affected; the highest degree of severity was observed in placentas with late resorptions. Placental degeneration correlated with a severe decline in maternal serum estradiol concentration. Supplementation with 0.5 and 1.0 μg of the synthetic estrogen estradiol cyclopentylpropionate per day reduced the severity of the degeneration in placentas with live fetuses. The present study demonstrates that both the placental degeneration and the increased incidence of late fetal resorptions are due to decreased levels of estrogen, since estrogen supplementation ameliorates the former and abolishes the latter. © 2013 Wiley Periodicals, Inc.

  9. Effects of estradiol and progesterone on the variability of the micronucleus assay

    International Nuclear Information System (INIS)

    Baeyens, Ans; Vandersickel, Veerle; Thierens, Hubert; Ridder, Leo De; Vral, Anne

    2005-01-01

    To investigate chromosomal radiosensitivity of lymphocytes the micronucleus (MN) assay has been used for many years. The results of these studies suggest the use of the MN assay as a biomarker for cancer predisposition. However, the MN assay has still some limitations associated with the reproducibility and sensitivity. Especially a high intra-individual variability has been observed. An explanation for this high intra-individual variability is not yet available. In literature it is suggested that the high variability among females is attributable to hormonal status. In this study we investigated if the high intra-individual variability in micronucleus formation in lymphocytes of females after in vitro exposure to ionising radiation is caused by variations in hormone levels of estradiol (E2) and progesterone (PROG). For this, the MN assay was performed on blood samples of 18 healthy women during 7 consecutive weeks while the estradiol and progesterone levels were determined at the same time. The MN assay was also examined in cultures of isolated blood lymphocytes with estradiol or progesterone levels added in vitro. The results demonstrated that estradiol and progesterone levels have no influence on the variations in radiation-induced MN yields observed in blood samples of healthy women. These conclusions were confirmed by the 'in vitro' experiments as no correlation between the MN yields and the concentrations of hormones (estradiol or progesterone) added in vitro to isolated lymphocytes cultures was observed

  10. Infrapatellar fat pad-derived stem cells maintain their chondrogenic capacity in disease and can be used to engineer cartilaginous grafts of clinically relevant dimensions.

    Science.gov (United States)

    Liu, Yurong; Buckley, Conor Timothy; Almeida, Henrique V; Mulhall, Kevin J; Kelly, Daniel John

    2014-11-01

    A therapy for regenerating large cartilaginous lesions within the articular surface of osteoarthritic joints remains elusive. While tissue engineering strategies such as matrix-assisted autologous chondrocyte implantation can be used in the repair of focal cartilage defects, extending such approaches to the treatment of osteoarthritis will require a number of scientific and technical challenges to be overcome. These include the identification of an abundant source of chondroprogenitor cells that maintain their chondrogenic capacity in disease, as well as the development of novel approaches to engineer scalable cartilaginous grafts that could be used to resurface large areas of damaged joints. In this study, it is first demonstrated that infrapatellar fat pad-derived stem cells (FPSCs) isolated from osteoarthritic (OA) donors possess a comparable chondrogenic capacity to FPSCs isolated from patients undergoing ligament reconstruction. In a further validation of their functionality, we also demonstrate that FPSCs from OA donors respond to the application of physiological levels of cyclic hydrostatic pressure by increasing aggrecan gene expression and the production of sulfated glycosaminoglycans. We next explored whether cartilaginous grafts could be engineered with diseased human FPSCs using a self-assembly or scaffold-free approach. After examining a range of culture conditions, it was found that continuous supplementation with both transforming growth factor-β3 (TGF-β3) and bone morphogenic protein-6 (BMP-6) promoted the development of tissues rich in proteoglycans and type II collagen. The final phase of the study sought to scale-up this approach to engineer cartilaginous grafts of clinically relevant dimensions (≥2 cm in diameter) by assembling FPSCs onto electrospun PLLA fiber membranes. Over 6 weeks in culture, it was possible to generate robust, flexible cartilage-like grafts of scale, opening up the possibility that tissues engineered using FPSCs

  11. Effects of chronic restraint stress and estradiol on open field activity, spatial memory, and monoaminergic neurotransmitters in ovariectomized rats.

    Science.gov (United States)

    Bowman, R E; Ferguson, D; Luine, V N

    2002-01-01

    Twenty-one days of chronic restraint stress impairs male rat performance on the radial arm maze [Luine et al. (1994) Brain Res. 639, 167-170], but enhances female rat performance [Bowman et al. (2001) Brain Res. 904, 279-289]. To assess possible ovarian hormone mechanisms underlying this sexually dimorphic response to stress, we examined chronic stress effects in ovariectomized rats. Ovariectomized rats received Silastic capsule implants containing cholesterol or estradiol and were assigned to a daily restraint stress (21 days, 6 h/day) or non-stress group. Following the stress period, subjects were tested for open field activity and radial arm maze performance. Stress and estradiol treatment affected open field activity. All stressed animals, with or without estradiol treatment, made fewer total outer sector crossings. In contrast, estradiol-treated animals, with or without stress, made more inner sector visits, an indication that estradiol decreased anxious behavior on the open field across time. As measured by the total number of visits required to complete the task, stress did not affect radial arm maze performance in ovariectomized rats, but estradiol-treated animals, with or without stress, performed better than non-treated animals on the radial arm maze. Stressed subjects receiving estradiol showed the best radial arm maze performance. Following killing, tissue samples were obtained from various brain regions known to contribute to learning and memory, and monoamine and metabolite levels were measured. Several changes were observed in response to both stress and estradiol. Most noteworthy, stress treatment decreased homovanillic acid levels in the prefrontal cortex, an effect not previously observed in stressed intact females. Estradiol treatment increased norepinephrine levels in CA3 region of the hippocampus, mitigating stress-dependent changes. Both stress and estradiol decreased dentate gyrus levels of 5-hydroxyindole acetic acid. In summary, the current

  12. Mineralocorticoid receptor haplotype, estradiol, progesterone and emotional information processing.

    Science.gov (United States)

    Hamstra, Danielle A; de Kloet, E Ronald; Quataert, Ina; Jansen, Myrthe; Van der Does, Willem

    2017-02-01

    Carriers of MR-haplotype 1 and 3 (GA/CG; rs5522 and rs2070951) are more sensitive to the influence of oral contraceptives (OC) and menstrual cycle phase on emotional information processing than MR-haplotype 2 (CA) carriers. We investigated whether this effect is associated with estradiol (E2) and/or progesterone (P4) levels. Healthy MR-genotyped premenopausal women were tested twice in a counterbalanced design. Naturally cycling (NC) women were tested in the early-follicular and mid-luteal phase and OC-users during OC-intake and in the pill-free week. At both sessions E2 and P4 were assessed in saliva. Tests included implicit and explicit positive and negative affect, attentional blink accuracy, emotional memory, emotion recognition, and risky decision-making (gambling). MR-haplotype 2 homozygotes had higher implicit happiness scores than MR-haplotype 2 heterozygotes (p=0.031) and MR-haplotype 1/3 carriers (pemotion recognition test than MR-haplotype 1/3 (p=0.001). Practice effects were observed for most measures. The pattern of correlations between information processing and P4 or E2 differed between sessions, as well as the moderating effects of the MR genotype. In the first session the MR-genotype moderated the influence of P4 on implicit anxiety (sr=-0.30; p=0.005): higher P4 was associated with reduction in implicit anxiety, but only in MR-haplotype 2 homozygotes (sr=-0.61; p=0.012). In the second session the MR-genotype moderated the influence of E2 on the recognition of facial expressions of happiness (sr=-0.21; p=0.035): only in MR-haplotype 1/3 higher E2 was correlated with happiness recognition (sr=0.29; p=0.005). In the second session higher E2 and P4 were negatively correlated with accuracy in lag2 trials of the attentional blink task (pemotional information processing. This moderating effect may depend on the novelty of the situation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Inclusion of 3H-estradiol-17#betta# in the chick embryo ovary in vitro

    International Nuclear Information System (INIS)

    Angelova, P.; Martinova, J.; K''ncheva, L.; Jordonov, Zh.; Bylgarska Akademiya na Naukite, Sofia)

    1982-01-01

    Basing on literature data on experimental investigation of genital differentiation of chick embryonal gonad in vitro, the authors have made their proposal that relationship between extragents and androgens in the favour of estradiol is of a great importance for differentiation of the gonad corti-- cal zone and for interruption of the meiosis process in cortical genital cells both genetically female and male (in the case of testis feminization). The autoradiographic investigation on 3 H-estradiol-17#betta# inclusion in an embryonal chick ovary in the period before the beginning of the meiotic prophase in genital cells has been performed in order to prove this hypothesis. The results obtained complement Gasc data on the presence of receptors for steroid hormones in embryonal chick gonads and confirm a conception that the development of indifferent gonad in female line is the same as the differentiation of cortical genital cells to oocyte conditioned by estradiol

  14. Male reproductive effects of octylphenol and estradiol in Fischer and Wistar rats

    DEFF Research Database (Denmark)

    Hossaini, Alireza; Dalgaard, Majken; Vinggaard, Anne

    2003-01-01

    to vehicle or 400 mg/kg bw of 4-tert-octylphenol administrated orally by gavage. Estradiol benzoate, at a dose of 40 mug/kg bw, was used as positive control agent. Treatment with estradiol benzoate decreased serum levels of testosterone, LH, FSH, inhibin and increased prolactin. Additionally, estradiol...... benzoate decreased the weight of all investigated reproductive organs, decreased sperm production and increased seminiferous tubular degeneration in both strains. More progressive effects on testis weight and histopathology were observed in the Fischer rats. Oral administration of octylphenol at 400 mg....../kg bw to both rat strains increased prolactin levels but had no effect on LH, FSH, testosterone or inhibin. In the octylphenol-treated Fischer rats the weights of the seminal vesicles and the levator ani/bulbocavernosus muscle were significantly decreased, whereas only the levator ani...

  15. Effects of 17β-estradiol on emissions of greenhouse gases in simulative natural water body.

    Science.gov (United States)

    Ruan, Aidong; Zhao, Ying; Liu, Chenxiao; Zong, Fengjiao; Yu, Zhongbo

    2015-05-01

    Environmental estrogens are widely spread across the world and are increasingly thought of as serious contaminators. The present study looks at the influence of different concentrations of 17β-estradiol on greenhouse gas emissions (CO2 , CH4 , and N2 O) in simulated systems to explore the relationship between environmental estrogen-pollution and greenhouse gas emissions in natural water bodies. The present study finds that 17β-estradiol pollution in simulated systems has significant promoting effects on the emissions of CH4 and CO2 , although no significant effects on N2 O emissions. The present study indicates that 17β-estradiol has different effects on the different elements cycles; the mechanism of microbial ecology is under review. © 2015 SETAC.

  16. Long-term estradiol treatment improves VIP-mediated vasodilation in atherosclerotic proximal coronary arteries

    DEFF Research Database (Denmark)

    Dalsgaard, T.; Mortensen, Alicja; Larsen, C. R.

    2003-01-01

    arteries. Female ovariectomized homozygous Watanabe heritable hyperlipidemic rabbits were randomized to 16 weeks treatment with 17beta-estradiol or placebo. The diet was semisynthetic, thereby avoiding the influence of phytoestrogens. Artery ring segments were mounted for isometric tension recordings...... in myographs. Following precontraction, the dose-response relationships for VIP and PACAP were evaluated. Treatment with 17beta-estradiol significantly improved the maximum VIP-mediated vasodilation (E-max, percentage of precontraction) in proximal coronary arteries (45.8 +/- 9.6% vs. 24.1 +/- 3.7%, p ....05). In the same artery segment, 17β-estradiol induced a significant decrease in the relative ratio between the repeated contractile response to potassium 30 and 120 mM (100 +/- 7% vs. 132 +/- 11%, p

  17. Negative effect of 17-beta-estradiol on growth parameters of goldfish (Carassius auratus

    Directory of Open Access Journals (Sweden)

    Reza Tarkhani

    2015-03-01

    Full Text Available Objective: To evaluate the effects of 17-beta-estradiol on growth factors of goldfish (Carassius auratus. Methods: To perform the test, 17-beta-estradiol was given 3 months period to fish at different doses as followed: control group, Group 1: 10 mg/kg food, Group 2: 25 mg/kg food and Group 3: 50 mg/kg food. For this purpose, a solution of hormone in pure ethanol used to spray on food. Feeding was done 3 times daily as an appetite. Comparing the mean values measured for length and weight using ANOVA. Results: Indicated with increase length and weight, the effects of the hormone get more distinct, so that with increase concentration of hormone, reduce weight and length. Conclusions: Estradiol along with testosterone and progesterone regulates final stages of oocyte maturation and ovulation. Various studies have proven the different concentrations of this hormone has different effects on the growth of different fishes.

  18. Intrinsic mechanism of estradiol-induced apoptosis in breast cancer cells resistant to estrogen deprivation.

    Science.gov (United States)

    Lewis, Joan S; Meeke, Kathleen; Osipo, Clodia; Ross, Eric A; Kidawi, Noman; Li, Tianyu; Bell, Eric; Chandel, Navdeep S; Jordan, V Craig

    2005-12-07

    We previously developed an estrogen receptor (ER)-positive breast cancer cell line (MCF-7:5C) that is resistant to long-term estrogen deprivation and undergoes rapid and complete apoptosis in the presence of physiologic concentrations of 17beta-estradiol. Here, we investigated the role of the mitochondrial apoptotic pathway in this process. Apoptosis in MCF-7:5C cells treated with estradiol, fulvestrant, or vehicle (control) was investigated by annexin V-propidium iodide double staining and 4',6-diamidino-2-phenylindole (DAPI) staining. Apoptosis was also analyzed in MCF-7:5C cells transiently transfected with small interfering RNAs (siRNAs) to apoptotic pathway components. Expression of apoptotic pathway intermediates was measured by western blot analysis. Mitochondrial transmembrane potential (psim) was determined by rhodamine-123 retention assay. Mitochondrial pathway activity was determined by cytochrome c release and cleavage of poly(ADP-ribose) polymerase (PARP) protein. Tumorigenesis was studied in ovariectomized athymic mice that were injected with MCF-7:5C cells. Differences between the treatment groups and control group were determined by two-sample t test or one-factor analysis of variance. All statistical tests were two-sided. MCF-7:5C cells treated with estradiol underwent apoptosis and showed increased expression of proapoptotic proteins, decreased psim, enhanced cytochrome c release, and PARP cleavage compared with cells treated with fulvestrant or vehicle. Blockade of Bax, Bim, and p53 mRNA expression by siRNA reduced estradiol-induced apoptosis relative to control by 76% [95% confidence interval (CI) = 73% to 79%, P estradiol-induced apoptosis in long-term estrogen-deprived breast cancer cells. Physiologic concentrations of estradiol could potentially be used to induce apoptosis and tumor regression in tumors that have developed resistance to aromatase inhibitors.

  19. In vivo measurement of tumor estradiol and Vascular Endothelial Growth Factor in breast cancer patients

    International Nuclear Information System (INIS)

    Garvin, Stina; Dabrosin, Charlotta

    2008-01-01

    Angiogenesis, crucial for tumor progression, is a process regulated in the tissue micro-environment. Vascular endothelial growth factor (VEGF) is a potent stimulatory factor of angiogenesis and a negative prognostic indicator of breast cancer. VEGF is biologically active in the extracellular space and hitherto, there has been a lack of techniques enabling sampling of angiogenic molecules such as VEGF in situ. The majority of breast cancers are estrogen-dependent, and estrogen has been shown to regulate VEGF in normal breast tissue and experimental breast cancer. We investigated if microdialysis may be applicable in human breast cancer for sampling of extracellular VEGF in situ and to explore if there is an association with local estradiol and VEGF levels in normal and cancerous breast tissue. Microdialysis was used to sample VEGF and estradiol in tumors and adjacent normal breast tissue in postmenopausal breast cancer patients. VEGF and estradiol were also measured in plasma, and immunohistochemical staining for VEGF was performed on tumor sections. We show that in vivo levels of extracellular VEGF were significantly higher in breast cancer tumors than in normal adjacent breast tissue. There was a significant positive correlation between estradiol and extracellular VEGF in normal breast tissue. However, no correlation was detected between estradiol and VEGF in tumors or between tumor VEGF and plasma VEGF. We conclude that VEGF and estradiol correlates significantly in normal breast tissue. Microdialysis may be used to provide novel insight in breast tumor biology and the regulation of molecules in the extracellular space of human breast tumors in vivo

  20. Evidence that the [3H]estradiol-binding protein in pancreas is localized in exocrine cells

    International Nuclear Information System (INIS)

    Grossman, A.; Richardson, S.B.; Altszuler, N.; Lane, B.

    1985-01-01

    Extracts of rat pancreas contain significant amounts of an [ 3 H]estradiol-binding protein. The amount of steroid-binding activity that could be measured varied considerably depending on the tonicity of the homogenizing medium. High speed supernatants of homogenates initially prepared in isotonic buffer contained about 10% of the binding activity as homogenates prepared in hypotonic buffer. Extraction with hypotonic buffer of pellets obtained by the isotonic procedure yielded most of the remaining [ 3 H]estradiol-binding activity. In an attempt to avoid errors resulting from incomplete homogenization and to detect possible changes in intracellular distribution of [ 3 H]estradiol-binding activity, pancreata were initially homogenized in isotonic buffer and centrifuged at high speed (100,000 X g; 1 hr). The pellet was then extracted with hypotonic buffer and centrifuged again at high speed, and both supernatants were analyzed for [ 3 H]estradiol-binding and amylase activities. Two or 14 days after treatment of male rats with streptozotocin, no apparent decline or redistribution of [ 3 H]estradiol-binding activity to the cytosol was noted despite extensive alteration of beta-islet cells, as determined by electron microscopic examination of sections of these pancreata and significant loss of insulin, as measured by RIA. Amylase activity was unaffected 2 days after streptozotocin treatment, but was depressed to about 1% of control levels at 14 days. Administration of insulin to the latter group of animals resulted in return of amylase to normal levels and a modest increase (approximately 50%) in [ 3 H]estradiol-binding activity

  1. A rapid enhancement of locomotor sensitization to amphetamine by estradiol in female rats.

    Science.gov (United States)

    Zovkic, Iva B; McCormick, Cheryl M

    2017-11-14

    Estradiol moderates the effects of drugs of abuse in both humans and rodents. Estradiol's enhancement of behavioral effects resulting from high (>2.5mg/kg) doses of amphetamine is established in rats; there is less evidence for the role of estradiol in locomotor effects elicited by lower doses, which are less aversive, increase incentive motivation, involve different neural mechanisms than higher doses, and often more readily reveal group differences than do higher doses. Further, the extent to which estradiol is required for the induction versus the expression of sensitization is unknown. To establish a protocol, we replicated the effects of estradiol on locomotor sensitization to amphetamine reported in a previous study that involved a high locomotor-activating dose (1.5mg/kg) of amphetamine, but with a lower dose. Ovariectomized female rats received 5μg of estradiol benzoate (EB) or OIL 30min before each of 5 treatments of 1.0mg/kg amphetamine or saline; all received a 0.5mg/kg challenge dose three days later. Compared with results for OIL, EB enhanced the locomotor-activating effects of repeated 1.0mg/kg amphetamine across treatment days. In contrast, on challenge day, there was no difference between EB-saline and EB-amphetamine to the lower dose (i.e., no sensitization). Experiments 2 and 3 involved a shorter induction (2days) and a lengthier withdrawal (9days) before the challenge test for the expression of sensitization to better differentiate the induction phase from the expression phase. In Expt2, EB-, and not OIL-, treated rats showed sensitization to 0.5mg/kg amphetamine; neither group showed sensitization to 1.5mg/kg amphetamine (ceiling effect?). In Expt3, rats were treated with EB either in both the induction and expression phases, in one of the phases only, or in neither phase. There was an effect of hormone treatment on challenge day and not on induction day; rats given EB on Challenge day showed sensitization to 0.5mg/kg amphetamine; OIL rats did

  2. Estrogen action in the mouse uterus: differential nuclear localization of estradiol in uterine cell types

    International Nuclear Information System (INIS)

    Korach, K.S.; Lamb, J.C.

    1981-01-01

    Autoradiographic studies of labeled steroid uptake in mouse uterine tissue indicated that labeled estradiol was predominantly sequestered in the nuclei of stromal and glandular epithelial cells at 1 h. Luminal epithelial cells did not show appreciable nuclear accumulation of labeled estradiol until 7-8 h after hormone injection. Studies using non-target tissues and unlabeled steroids indicated that the nuclear uptake events were tissue and estrogen steroid specific. The temporal pattern of steroid hormone uptake in the uterus would suggest that an initial interaction in stromal and glandular epithelial cells may be required prior to nuclear stimulation in the luminal epithelial target cell

  3. Estradiol treatment in preadolescent females enhances adolescent spatial memory and differentially modulates hippocampal region-specific phosphorylated ERK labeling.

    Science.gov (United States)

    Wartman, Brianne C; Keeley, Robin J; Holahan, Matthew R

    2012-10-24

    Estrogen levels in rats are positively correlated with enhanced memory function and hippocampal dendritic spine density. There is much less work on the long-term effects of estradiol manipulation in preadolescent rats. The present work examined how injections of estradiol during postnatal days 19-22 (p19-22; preadolescence) affected water maze performance and hippocampal phosphorylated ERK labeling. To investigate this, half of the estradiol- and vehicle-treated female rats were trained on a water maze task 24h after the end of estradiol treatment (p23-27) while the other half was not trained. All female rats were tested on the water maze from p40 to p44 (adolescence) and hippocampal pERK1/2 labeling was assessed as a putative marker of neuronal plasticity. During adolescence, preadolescent-trained groups showed lower latencies than groups without preadolescent training. Retention data revealed lower latencies in both estradiol groups, whether preadolescent trained or not. Immunohistochemical detection of hippocampal pERK1/2 revealed elevations in granule cell labeling associated with the preadolescent trained groups and reductions in CA1 labeling associated with estradiol treatment. These results show a latent beneficial effect of preadolescent estradiol treatment on adolescent spatial performance and suggest an organizational effect of prepubescent exogenously applied estradiol. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  4. Butyl paraben and propyl paraben modulate bisphenol A and estradiol concentrations in female and male mice

    Energy Technology Data Exchange (ETDEWEB)

    Pollock, Tyler; Weaver, Rachel E.; Ghasemi, Ramtin; Catanzaro, Denys de, E-mail: decatanz@mcmaster.ca

    2017-06-15

    People are routinely exposed to the antimicrobial preservatives butyl paraben (BP) and propyl paraben (PP), as well as the monomer of polycarbonate plastics, bisphenol A (BPA). These chemicals are reliably detected in human urine and potentially interact. We investigated whether BP or PP exposure can modulate the concentrations of {sup 14}C-BPA and 17β-estradiol (E{sub 2}). Female and male CF1 mice were each given a subcutaneous injection of oil containing 0 (vehicle), 1, 3, or 9 mg BP or PP, then given a dietary supplement containing 50 μg/kg {sup 14}C-BPA. Radioactivity was measured in tissues through liquid scintillation counting. Significantly elevated {sup 14}C-BPA concentrations were observed following BP treatment in blood serum of both sexes, as well as the lungs, uterus, and ovaries of females and the testes and epididymides of males. Treatment with PP significantly elevated {sup 14}C-BPA concentrations in the uterus only. In another experiment, female and male CF1 mice were each injected with vehicle, 3 mg BP, or 3 mg PP, and E{sub 2} was measured in urine 2–12 h later. Whereas PP did not affect E{sub 2}, BP significantly elevated E{sub 2} 6–10 h after injection in females and 8 h after injection in males. These data indicate that BP and PP can alter the pharmacokinetics of BPA in vivo, and that BP can modulate E{sub 2} concentrations. These results are consistent with evidence that parabens inhibit enzymes that are critical for BPA and E{sub 2} metabolism, and demonstrate the importance of considering concurrent exposure to multiple chemicals when determining regulatory exposure limits. - Highlights: • We studied whether paraben exposure affects the distribution of oral {sup 14}C-BPA. • Elevated {sup 14}C–BPA was observed in mice given butyl or propyl paraben. • We also studied whether paraben exposure affects natural E{sub 2} levels in urine. • Elevated E{sub 2} was observed in mice given butyl, but not propyl, paraben. • Parabens may

  5. Fluoxetine ameliorates cartilage degradation in osteoarthritis by inhibiting Wnt/β-catenin signaling.

    Science.gov (United States)

    Miyamoto, Kentaro; Ohkawara, Bisei; Ito, Mikako; Masuda, Akio; Hirakawa, Akihiro; Sakai, Tadahiro; Hiraiwa, Hideki; Hamada, Takashi; Ishiguro, Naoki; Ohno, Kinji

    2017-01-01

    Abnormal activation of the Wnt/β-catenin signaling is implicated in the osteoarthritis (OA) pathology. We searched for a pre-approved drug that suppresses abnormally activated Wnt/β-catenin signaling and has a potency to reduce joint pathology in OA. We introduced the TOPFlash reporter plasmid into HCS-2/8 human chondrosarcoma cells to estimate the Wnt/β-catenin activity in the presence of 10 μM each compound in a panel of pre-approved drugs. We found that fluoxetine, an antidepressant in the class of selective serotonin reuptake inhibitors (SSRI), down-regulated Wnt/β-catenin signaling in human chondrosarcoma cells. Fluoxetine inhibited both Wnt3A- and LiCl-induced loss of proteoglycans in chondrogenically differentiated ATDC5 cells. Fluoxetine increased expression of Sox9 (the chondrogenic master regulator), and decreased expressions of Axin2 (a marker for Wnt/β-catenin signaling) and Mmp13 (matrix metalloproteinase 13). Fluoxetine suppressed a LiCl-induced increase of total β-catenin and a LiCl-induced decrease of phosphorylated β-catenin in a dose-dependent manner. An in vitro protein-binding assay showed that fluoxetine enhanced binding of β-catenin with Axin1, which is a scaffold protein forming the degradation complex for β-catenin. Fluoxetine suppressed LiCl-induced β-catenin accumulation in human OA chondrocytes. Intraarticular injection of fluoxetine in a rat OA model ameliorated OA progression and suppressed β-catenin accumulation.

  6. Vitamin A family compounds, estradiol, and docetaxel in proliferation, apoptosis and immunocytochemical profile of human ovary endometrioid cancer cell line CRL-11731.

    Directory of Open Access Journals (Sweden)

    Dorota Lemancewicz

    2010-01-01

    Full Text Available Endometrioid carcinoma represents approximately 10% of cases of the malignant ovarian epithelial tumors. According to literature, the vitamin A (carotenoids and retinoids plays an essential role in cell proliferation, differentiation and apoptosis in both normal and neoplastic ovarian tissues. Apart from that, the retinoids alter a cytotoxic effect of chemiotherapeutics, i.e. docetaxel, on ovarian cancer cell lines. Retinoids act on cancer cells throughout different mechanism than taxanes, so they may be the potential candidates for the new treatment strategies of ovarian cancer. The aim of the study was to determine the effects of vitamin A family compounds (retinol, beta-carotene, lycopene, all-trans -, 9-cis - and 13-cis retinoic acid on the growth and proliferation of CRL-11731 endometrioid ovary cancer cell line and on docetaxel and estradiol activity in this culture. The assay was based on [3H] thymidine incorporation and the proliferative activity of PCNA- and Ki 67-positive cells. The apoptotic index and expression of the Bcl-2 and p53 antigens in CRL-11731 cells were also studied. Among vitamin A family compounds retinol and carotenoids, but not retinoids, inhibited the growth of cancer cells in dose dependent manner. Only the concentration of 100 muM of docetaxel inhibited incorporation [3H] thymidine into CRL-11731 cancer cells. Retinol (33.4%+/-8.5, carotenoids (beta-carotene 20 muM 4.7%+/-2.9, 50 muM 2.2%+/-0.9; lycopene 10 muM 7.6%+/-0.8, 20 muM 5.2%+/-2.5, 50 muM 2.9%+/-1.2, and 13-cis retinoic acid (19.7%+/-2.2 combined with docetaxel (100 muM significantly decreased the percentage of proliferating cells (p<0.0001. The antiproliferative action of lycopene alone and in combination with docetaxel was also confirmed in immunohistochemical examination (decreased the percentage of PCNA and Ki67 positive cells. Also retinol (10 muM and lycopene (20 and 50 muM combined with estradiol (0.01 muM statistically decreased the percentage of

  7. Morphological Differentiation Towards Neuronal Phenotype of SH-SY5Y Neuroblastoma Cells by Estradiol, Retinoic Acid and Cholesterol.

    Science.gov (United States)

    Teppola, Heidi; Sarkanen, Jertta-Riina; Jalonen, Tuula O; Linne, Marja-Leena

    2016-04-01

    Human SH-SY5Y neuroblastoma cells maintain their potential for differentiation and regression in culture conditions. The induction of differentiation could serve as a strategy to inhibit cell proliferation and tumor growth. Previous studies have shown that differentiation of SH-SY5Y cells can be induced by all-trans-retinoic-acid (RA) and cholesterol (CHOL). However, signaling pathways that lead to terminal differentiation of SH-SY5Y cells are still largely unknown. The goal of this study was to examine in the RA and CHOL treated SH-SY5Y cells the additive impacts of estradiol (E2) and brain-derived neurotrophic factor (BDNF) on cell morphology, cell population growth, synaptic vesicle recycling and presence of neurofilaments. The above features indicate a higher level of neuronal differentiation. Our data show that treatment for 10 days in vitro (DIV) with RA alone or when combined with E2 (RE) or CHOL (RC), but not when combined with BDNF (RB), significantly (p differentiation.

  8. [β-estradiol activates BK(Ca) in mesenteric artery smooth muscle cells of post-menopause women].

    Science.gov (United States)

    Cheng, Jun; Zeng, Xiao-Rong; Li, Peng-Yun; Lu, Ting-Ting; Tan, Xiao-Qiu; Wen, Jing; Yang, Yan

    2012-04-25

    The aim of the present study was to study the effect of β-estradiol (β-E(2)) on the large-conductance Ca(2+)-activated potassium (BK(Ca)) channel in mesenteric artery smooth muscle cells (SMCs). The mesenteric arteries were obtained from post-menopause female patients with abdominal surgery, and the SMCs were isolated from the arteries using an enzymatic disassociation. According to the sources, the SMCs were divided into non-hypertension (NH) and essential hypertension (EH) groups. Single channel patch clamp technique was used to investigate the effect of β-E(2) and ICI 182780 (a specific blocker of estrogen receptor) on BK(Ca) in the SMCs. The results showed the opening of BK(Ca) in the SMCs was voltage and calcium dependent, and could be blocked by IbTX. β-E(2) (100 μmol/L) significantly increased open probability (Po) of BK(Ca) in both NH and EH groups. After β-E(2) treatment, NH group showed higher Po of BK(Ca) compared with EH group. ICI 182780 could inhibit the activating effect of β-E(2) on BK(Ca) in no matter NH or EH groups. These results suggest β-E(2) activates BK(Ca) in mesenteric artery SMCs from post-menopause women via estrogen receptor, but hypertension may decline the activating effect of β-E(2) on BK(Ca).

  9. 17β-Estradiol Protects Mitochondrial Functions through Extracellular-Signal-Regulated Kinase in C2C12 Muscle Cells

    Directory of Open Access Journals (Sweden)

    Ana C. Ronda

    2013-10-01

    Full Text Available Background/Aims: We have previously shown that exposure to 17β-estradiol (E2 prior to induction of apoptosis with H2O2 protects skeletal muscle cells against oxidative damage. However, the mechanism involved in the protective action of the hormone is poorly understood. In the present study, we focused on the mechanism by which ERK mediates this survival effect in connection with COXIV activity and mitochondrial membrane potential. Methods: Immunocytochemistry, Western blot, cytochrome c oxidase complex IV (COXIV activity, coimmunoprecipitation and JC-1 dye by flow cytometry were carried out using C2C12 myoblasts as experimental model. Results: E2 is able to activate ERK and then induces its translocation to mitochondria. Using the pharmacological inhibitor of ERK activation U0126 we show that E2, through ERK activation, is able to enhance COXIV activity. Moreover, the hormone increases the interaction between COXIV and ERK. Also, we found that hydrogen peroxide decreases COXIV activity and that preincubation of the cells with E2 prior to induction of apoptosis prevents this effect. In addition, we observe that the estrogen inhibits the collapse of mitochondrial membrane potential induced by H2O2, involving ERK and COXIV. Conclusion: Our data demonstrate that E2 promotes ERK activation and translocation to mitochondria preventing the decline in COXIV activity and in turn, alteration of mitochondrial membrane potential by oxidative stress, in C2C12 myoblasts.

  10. Proteome-wide effect of 17-β-Estradiol and Lipoxin A4 in an endometriotic epithelial cell line

    Directory of Open Access Journals (Sweden)

    Jonathan eSobel

    2016-01-01

    Full Text Available Endometriosis affects approximately 10% of the women of reproductive age. This chronic gynecological inflammatory disease results in a decreased quality of life for patients, with the main symptoms including chronic pelvic pain and infertility. The steroid hormone 17-β Estradiol (E2 plays a key role in the pathology. Our previous studies showed that the anti-inflammatory lipid Lipoxin A4 (LXA4 acts an estrogen receptor alpha agonist in endometrial epithelial cells, inhibiting certain E2-mediated effects. LXA4 also prevents the progression of endometriosis in a mouse model via anti-proliferative mechanisms and by impacting mediators downstream of ER signaling. The aim of the present study was therefore to examine global proteomic changes evoked by E2 and LXA4 in endometriotic epithelial cells. E2 impacted a greater number of proteins in endometriotic epithelial cells than LXA4. Interestingly, the combination of E2 and LXA4 resulted in a reduced number of regulated proteins, with LXA4 mediating a suppressive effect on E2-mediated signaling. These proteins are involved in diverse pathways of relevance to endometriosis pathology and metabolism, including mRNA translation, growth, proliferation, proteolysis and immune responses. In summary, this study sheds light on novel pathways involved in endometriosis pathology and furthers understanding of signaling pathways activated by estrogenic molecules in endometriotic epithelial cells.

  11. Neuroprotective effects of 17β-estradiol in a rat model of neonatal X radiation

    International Nuclear Information System (INIS)

    Caceres, L.G.; Aon, L.; Saraceno, E.; Capani, F.; Guelman, L.R.

    2009-01-01

    Developing Central Nervous System (CNS) is vulnerable to radiation-induced reactive oxygen species (ROS). The consequent oxidative stress has been shown to produce changes at behavioral, biochemical and histological levels in cerebellum (CE) and hippocampus (HIP). The aim of the present work was to test if 17β-estradiol, a potential neuroprotector, was able to counteract these changes. Neonatal male Wistar rats were X-irradiated (5 Gy) in their cephalic ends up to 48hs of postnatal life and a group of this animals was treated with 17β-estradiol (5 g/g). Open field (OF) test, ROS levels, as well as a histological assessment, were performed at 30 postnatal days. Administration of 17β-estradiol improved the short-term habituation and decreased the time spent in the centre in the OF. ROS levels returned to control in HIP and the cytoarchitecture of CE was reconstituted. These results suggest that 17β-estradiol was able to counteract the effects of X-rays at behavioral, biochemical and histological levels, probably acting through an antioxidant mechanism. (authors)

  12. Differential effects of testosterone, dihydrotestosterone and estradiol on carotenoid deposition in an avian sexually selected signal

    NARCIS (Netherlands)

    Casagrande, Stefania; Dijkstra, Cor; Tagliavini, James; Goerlich, Vivian C.; Groothuis, Ton G. G.

    Recent studies have demonstrated that carotenoid-based traits are under the control of testosterone (T) by up-regulation of carotenoid carriers (lipoproteins) and/or tissue-specific uptake of carotenoids. T can be converted to dihydrotestosterone (DHT) and estradiol (E2), and variation in conversion

  13. Pattern recognition of estradiol, testosterone and dihydrotestosterone in children's saliva samples using stochastic microsensors

    NARCIS (Netherlands)

    Stefan-van Staden, R.I.; Gugoaşă, L.A.; Calenic, B.; Legler, J.

    2014-01-01

    Stochastic microsensors based on diamond paste and three types of electroactive materials (maltodextrin (MD), α-cyclodextrin (α-CD) and 5,10,15,20-tetraphenyl-21H,23H porphyrin (P)) were developed for the assay of estradiol (E2), testosterone (T2) and dihydrotestosterone (DHT) in children's saliva.

  14. Effects of bromocriptine on [3H]estradiol binding in cytosol of anterior pituitary

    International Nuclear Information System (INIS)

    De Nicola, A.F.; Weisenberg, L.S.; Arakelian, M.C.; Libertun, C.

    1981-01-01

    The hypothalamus may control hormone receptors in the anterior pituitary either by a direct trophic effect or indirectly by regulation of serum pituitary hormone levels. Rats whose medial basal hypothalamus had been destroyed in order to suppress neural control of the gland showed a reduction in [ 3 H]estradiol binding in the anterior pituitary and high serum PRL levels; both changes were reversed by treatment of the lesioned rats with daily injections of bromocriptine, a dopamine agonist. In nonlesioned animals, the same treatment did not modify significantly those parameters. In another hyperprolactinemic model (rats with anterior pituitaries transplanted under the kidney capsule), [ 3 H]estradiol binding by the in situ pituitaries of the host rats was similar to that in the nongrafted controls. These results suggest that changes due to median eminence lesion are reversible and that bromocriptine is able to act as a substitutive therapy which restores binding of estradiol in glands whose receptors have been decreased by the effect of the lesion. High PRL levels due to pituitary transplant do not account for the observed changes in the pituitary estradiol binding

  15. Identification of target cells by immunohistochemical detection of covalently rearranged estradiol in rehydrated paraffin sections.

    Science.gov (United States)

    Jungblut, P W; Sierralta, W D

    1998-04-01

    Estradiol is released from the binding niche of the receptor and covalently arrested in the molecular vicinity by the Mannich reaction during target fixation in acetic acid/formaldehyde. The exposed steroid is freely accessible for appropriate antibodies. It can be visualized in sections by the second antibody/enzyme technique in high resolution and without enhancements.

  16. Cortisol Interferes with the Estradiol-Induced Surge of Luteinizing Hormone in the Ewe1

    Science.gov (United States)

    Wagenmaker, Elizabeth R.; Breen, Kellie M.; Oakley, Amy E.; Pierce, Bree N.; Tilbrook, Alan J.; Turner, Anne I.; Karsch, Fred J.

    2008-01-01

    Two experiments were conducted to test the hypothesis that cortisol interferes with the positive feedback action of estradiol that induces the luteinizing hormone (LH) surge. Ovariectomized sheep were treated sequentially with progesterone and estradiol to create artificial estrous cycles. Cortisol or vehicle (saline) was infused from 2 h before the estradiol stimulus through the time of the anticipated LH surge in the artificial follicular phase of two successive cycles. The plasma cortisol increment produced by infusion was ∼1.5 times greater than maximal concentrations seen during infusion of endotoxin, which is a model of immune/inflammatory stress. In experiment 1, half of the ewes received vehicle in the first cycle and cortisol in the second; the others were treated in reverse order. All ewes responded with an LH surge. Cortisol delayed the LH surge and reduced its amplitude, but both effects were observed only in the second cycle. Experiment 2 was modified to provide better control for a cycle effect. Four treatment sequences were tested (cycle 1-cycle 2): vehicle-vehicle, cortisol-cortisol, vehicle-cortisol, cortisol-vehicle. Again, cortisol delayed but did not block the LH surge, and this delay occurred in both cycles. Thus, an elevation in plasma cortisol can interfere with the positive feedback action of estradiol by delaying and attenuating the LH surge. PMID:19056703

  17. PTHrP potentiating estradiol-induced vitellogenesis in sea bream (Sparus auratus, L.)

    NARCIS (Netherlands)

    Bevelander, G.S.; Hang, X.; Abbink, W.; Spanings, F.A.T.; Canario, A.V.; Flik, G.

    2006-01-01

    In fish, vitellogenin is an important nutritional precursor protein produced solely in the liver and released into the blood where it binds calcium. In the gilthead sea bream (Sparus auratus) 17beta-Estradiol (E2) plays an important role in the synthesis of vitellogenin, but also the pituitary

  18. Estradiol Therapy After Menopause Mitigates Effects of Stress on Cortisol and Working Memory.

    Science.gov (United States)

    Herrera, Alexandra Ycaza; Hodis, Howard N; Mack, Wendy J; Mather, Mara

    2017-12-01

    Postmenopausal estradiol therapy (ET) can reduce the stress response. However, it remains unclear whether such reductions can mitigate effects of stress on cognition. Investigate effects of ET on cortisol response to a physical stressor, cold pressor test (CPT), and whether ET attenuates stress effects on working memory. Women completed the CPT or control condition across two sessions and subsequently completed a sentence span task. General community: Participants were recruited from the Early vs Late Intervention Trial with Estradiol (ELITE). ELITE participants (mean age = 66, standard deviation age = 6.8) in this study did not suffer from any major chronic illness or use medications known to affect the stress response or cognition. Participants had received a median of randomized 4.7 years of estradiol (n = 21) or placebo (n = 21) treatment at time of participation in this study. Salivary cortisol and sentence span task performance. Women assigned to estradiol exhibited blunted cortisol responses to CPT compared with placebo (P = 0.017) and lesser negative effects of stress on working memory (P = 0.048). We present evidence suggesting ET may protect certain types of cognition in the presence of stress. Such estrogenic protection against stress hormone exposure may prove beneficial to both cognition and the neural circuitry that maintains and propagates cognitive faculties. Copyright © 2017 Endocrine Society

  19. Simultaneous determination of ethinyl estradiol and drospirenone in oral contraceptive by high performance liquid chromatography

    Directory of Open Access Journals (Sweden)

    Viviane Benevenuti Silva

    2013-09-01

    Full Text Available A simple, rapid, economical and reliable high performance liquid chromatographic method has been developed and successfully applied in simultaneous determination of ethinyl estradiol and drospirenone in coated tablets. The HPLC method was performed on a LiChroCART® 100RP column (125x4 mm i.d., 5 µm with acetonitrile:water 50:50 (v/v as mobile phase, pumped at a flow rate of 1.0 mL.min-1. The fluorescence detection for ethinyl estradiol was made at λex= 280 nm and λem= 310 nm and a UV detection for drospirenone was made at 200 nm. The elution time for ethinyl estradiol and drospirenone were 4.0 and 5.7 min, respectively. The method was validated in accordance to USP 34 guidelines. The proposed HPLC method presented advantages over reported methods and is suitable for quality control assays of ethinyl estradiol and drospirenone in coated tablets.

  20. The influence of elevated preovulatory estradiol on apoptosis in the trophectoderm of day 16 bovine conceptuses

    Science.gov (United States)

    Preovulatory estradiol impacts follicular growth, oocyte maturation, sperm transport, uterine environment, and embryo survival/development. It has also been reported that cows in standing estrus within 24 hours of fixed-time AI have greater pregnancy success compared to cows that do not exhibit stan...

  1. Effects of Estradiol on Learned Helplessness and Associated Remodeling of Hippocampal Spine Synapses in Female Rats

    Science.gov (United States)

    Hajszan, Tibor; Szigeti-Buck, Klara; Sallam, Nermin L; Bober, Jeremy; Parducz, Arpad; MacLusky, Neil J; Leranth, Csaba; Duman, Ronald S

    2009-01-01

    Background Despite the fact that women are twice as likely to develop depression as men, our understanding of depression neurobiology in females is limited. We have recently reported in male rats that development of helpless behavior is associated with a severe loss of hippocampal spine synapses, which is reversed by treatment with the antidepressant, desipramine. Considering the fact that estradiol has a hippocampal synaptogenic effect similar to those of antidepressants, the presence of estradiol during the female reproductive life may influence behavioral and synaptic responses to stress and depression. Methods Using electron microscopic stereology, we analyzed hippocampal spine synapses in association with helpless behavior in ovariectomized female rats (n=70), under different conditions of estradiol exposure. Results Stress induced an acute and persistent loss of hippocampal spine synapses, while subchronic treatment with desipramine reversed the stress-induced synaptic loss. Estradiol supplementation given either prior to stress or prior to escape testing of nonstressed animals both increased the number of hippocampal spine synapses. Correlation analysis demonstrated a statistically significant negative correlation between the severity of helpless behavior and hippocampal spine synapse numbers. Conclusions These findings suggest that hippocampal spine synapse remodeling may be a critical factor underlying learned helplessness and, possibly, the neurobiology of depression. PMID:19811775

  2. Lipid profiling and transcriptomic analysis reveals a functional interplay between estradiol and growth hormone in liver

    DEFF Research Database (Denmark)

    Fernández-Pérez, Leandro; Santana-Farré, Ruymán; Mirecki-Garrido, Mercedes de

    2014-01-01

    17β-estradiol (E2) may interfere with endocrine, metabolic, and gender-differentiated functions in liver in both females and males. Indirect mechanisms play a crucial role because of the E2 influence on the pituitary GH secretion and the GHR-JAK2-STAT5 signaling pathway in the target tissues. E2,...

  3. Determination of urinary estradiol using an enzymatic method during the menstrual cycle

    International Nuclear Information System (INIS)

    Patricot, M.C.; Mathian, B.; Serpentie, S.; Revol, A.

    1986-01-01

    The aim of this study was to compare the results of enzymatic determination of urinary estradiol with results from a method using isotope dilution-mass fragmentography. Urine samples were collected from women during the menstrual cycle. The results obtained differed in absolute values, but showed good correlation. (Auth.)

  4. 17ß-Estradiol Is Necessary for Extinction of Cocaine Seeking in Female Rats

    Science.gov (United States)

    Twining, Robert C.; Tuscher, Jennifer J.; Doncheck, Elizabeth M.; Frick, Karyn M.; Mueller, Devin

    2013-01-01

    Human and preclinical models of addiction demonstrate that gonadal hormones modulate acquisition of drug seeking. Little is known, however, about the effects of these hormones on extinction of drug-seeking behavior. Here, we investigated how 17ß-estradiol (E[subscript 2]) affects expression and extinction of cocaine seeking in female rats. Using a…

  5. Synthesis and evaluation of a new series of 17α-[123I]iodovinyl estradiols

    International Nuclear Information System (INIS)

    Kabalka, George W.; Shoup, Timothy M.; Daniel, Gregory B.; Goodman, Mark M.

    2000-01-01

    A series of 17α-substituted estradiols was synthesized in which the stereochemical characteristics of carbons 20 and 21 were modified. It was found that the (Z)-isomer demonstrated more favorable receptor binding affinity than the corresponding (E)-isomer

  6. Modulating Action of 17β Estradiol on Urinary Volume and Renal ...

    African Journals Online (AJOL)

    This study determined the effect of combined administration of 17β estradiol and dietary salt on some renal parameters. Thirty-two female Albino rats were used for this study and they were assigned into four groups consist of eight rats in each group. The group A served as control while groups B, C and D were given daily ...

  7. Sexual Function in Women on Estradiol or Venlafaxine for Hot Flushes: A Randomized Controlled Trial

    Science.gov (United States)

    Reed, Susan D.; Mitchell, Caroline M.; Joffe, Hadine; Cohen, Lee; Shifren, Jan L.; Newton, Katherine M.; Freeman, Ellen W.; Larson, Joseph C.; Manson, JoAnn E.; LaCroix, Andrea Z.; Guthrie, Katherine A.

    2014-01-01

    Objective To evaluate sexual function in midlife women taking low-dose oral estradiol or venlafaxine for hot flushes. Methods In an 8-week randomized controlled trial among women aged 40-62 years, sexual function was compared between oral estradiol 0.5 mg/day or venlafaxine 75 mg/day (both compared with placebo). Measures included composite and 6 domain scores from the Female Sexual Function Index (FSFI) and sexually related personal distress. Results Participants were aged 54.6 (standard deviation [SD] 3.8) years, 59% Caucasian, with 8.1 (SD 5.3) daily hot flushes. Median composite baseline FSFI score was 16.3 (SD 11.9, n=256) for all women and 21.7 (SD 9.3, n=198) among sexually active women. Composite mean FSFI change from baseline to week-8 was 1.4 (95% Confidence Interval [CI] -0.4, 3.2) for estradiol, 1.1 (95% CI -0.5, 2.7) for venlafaxine and -0.3 (95% CI -1.6, 1.0) for placebo. Composite FSFI and sexually-related distress change from baseline did not differ between estradiol and placebo (p= 0.38, p=0.30) or venlafaxine and placebo (p=0.79, p=0.48). Among sexually active women, FSFI domain score change from baseline differences (active compared with placebo) in desire was 0.3 (95% CI 0.0, 0.6) for estradiol; -0.6 (95% CI -1.2, 0.0) in orgasm for venlafaxine, and 0.9 (95% CI 0.2, 1.6) in penetration pain for venlafaxine. No women reported adverse events related to sexual dysfunction. Conclusions Overall sexual function among nondepressed midlife women experiencing hot flushes did not change over 8-weeks with low-dose oral estradiol or venlafaxine (compared with placebo), although subtle increase in desire (estradiol), and decreases in orgasm and pain (venlafaxine) may exist. PMID:25004335

  8. Effects of estradiol and venlafaxine on insomnia symptoms and sleep quality in women with hot flashes.

    Science.gov (United States)

    Ensrud, Kristine E; Guthrie, Katherine A; Hohensee, Chancellor; Caan, Bette; Carpenter, Janet S; Freeman, Ellen W; LaCroix, Andrea Z; Landis, Carol A; Manson, JoAnn; Newton, Katherine M; Otte, Julie; Reed, Susan D; Shifren, Jan L; Sternfeld, Barbara; Woods, Nancy F; Joffe, Hadine

    2015-01-01

    Determine effects of low-dose estradiol and low-dose venlafaxine on self-reported sleep measures in menopausal women with hot flashes. 3-arm double-blind randomized trial. Participants assigned in a 2:2:3 ratio to 17β estradiol 0.5 mg/day (n = 97), venlafaxine XR 75 mg/day (n = 96), or placebo (n = 146) for 8 weeks. Academic research centers. 339 community-dwelling perimenopausal and postmenopausal women with ≥2 bothersome hot flashes per day. Insomnia symptoms (Insomnia Severity Index [ISI]) and sleep quality (Pittsburgh Sleep Quality Index [PSQI]) at baseline, week 4 and 8; 325 women (96%) provided ISI data and 312 women (92%) provided PSQI data at baseline and follow-up. At baseline, mean (SD) hot flash frequency was 8.1/day (5.3), mean ISI was 11.1 (6.0), and mean PSQI was 7.5 (3.4). Mean (95% CI) change from baseline in ISI at week 8 was -4.1 points (-5.3 to -3.0) with estradiol, -5.0 points (-6.1 to -3.9) with venlafaxine, and -3.0 points (-3.8 to -2.3) with placebo (P overall treatment effect vs. placebo 0.09 for estradiol and 0.007 for venlafaxine). Mean (95% CI) change from baseline in PSQI at week 8 was -2.2 points (-2.8 to -1.6) with estradiol, -2.3 points (-2.9 to -1.6) with venlafaxine, and -1.2 points (-1.7 to -0.8) with placebo (P overall treatment effect vs. placebo 0.04 for estradiol and 0.06 for venlafaxine). Among perimenopausal and postmenopausal women with hot flashes, both low dose oral estradiol and low-dose venlafaxine compared with placebo modestly reduced insomnia symptoms and improved subjective sleep quality. NCT01418209 at www.clinicaltrials.gov. © 2014 Associated Professional Sleep Societies, LLC.

  9. Investigation of testosterone, androstenone, and estradiol metabolism in HepG2 cells and primary culture pig hepatocytes and their effects on 17βHSD7 gene expression.

    Directory of Open Access Journals (Sweden)

    Gang Chen

    Full Text Available Steroid metabolism is important in various species. The accumulation of androgen metabolite, androstenone, in pig adipose tissue is negatively associated with pork flavor, odour and makes the meat unfit for human consumption. The 17β-hydroxysteroid dehydrogenase type 7 (17βHSD7 expressed abundantly in porcine liver, and it was previously suggested to be associated with androstenone levels. Understanding the enzymes and metabolic pathways responsible for androstenone as well as other steroids metabolism is important for improving the meat quality. At the same time, metabolism of steroids is known to be species- and tissue-specific. Therefore it is important to investigate between-species variations in the hepatic steroid metabolism and to elucidate the role of 17βHSD7 in this process. Here we used an effective methodological approach, liquid chromatography coupled with mass spectrometry, to investigate species-specific metabolism of androstenone, testosterone and beta-estradiol in HepG2 cell line, and pig cultured hepatocytes. Species- and concentration-depended effect of steroids on 17βHSD7 gene expression was also investigated. It was demonstrated that the investigated steroids can regulate the 17βHSD7 gene expression in HepG2 and primary cultured porcine hepatocytes in a concentration-dependent and species-dependent pattern. Investigation of steroid metabolites demonstrated that androstenone formed a 3'-hydroxy compound 3β-hydroxy-5α-androst-16-ene. Testosterone was metabolized to 4-androstene-3,17-dione. Estrone was found as the metabolite for β-estradiol. Inhibition study with 17βHSD inhibitor apigenin showed that apigenin didn't affect androstenone metabolism. Apigenin at high concentration (50 µM tends to inhibit testosterone metabolism but this inhibition effect was negligible. Beta-estradiol metabolism was notably inhibited with apigenin at high concentration. The study also established that the level of testosterone and β-estradiol

  10. Kombinasi Calcitriol dan Ethynil Ethyl Estradiol Meningkatkan Ekskresi Kalsium Urin dan Risiko Urolitiasis pada Tikus Ovariektomi

    Directory of Open Access Journals (Sweden)

    Hartiningsih Hartiningsih

    2017-06-01

    Full Text Available The high excretion of calcium (Ca in the urine can trigger the formation of urolith. Estrogen and calcitriol decrease urinary Ca excretion. This study aims to examine the combination of calcitriol and ethinyl ethyl estradiol against Ca urinary excretion and urolithiasis risk of ovariectomized rats. Twentyfive female Wistar rats eight weeks old were divided into five groups: i normal control (NK; ii ovariectomized control (OVK; iii ovariectomized + calcitriol (OVD; iv ovariectomized + ethinyl ethyl estradiol (OVE; and v ovariectomized + combination calcitriol and ethinyl ethyl estradiol (OVDE. Seven weeks post-ovariectomy, each rat was put in an individual metabolic cage for the study of Ca balance. At day 4 to 7 of the study, residual feed, urine, and feces were collected daily for Ca analysis. At day 8, the rats were euthanized, the left kidney were collected for histopathological examination. The results showed that combination of calcitriol and ethinyl ethyl estradiol in OVDE rats caused Ca intake and Ca intestinal absorption significantly higher, and urinary Ca excretion tended to be higher although not significantly different compared to OVK rats. Calcium excretion in OVK rat urine was higher compared to the NK rats. The kidney histopathological changes of OVK rats were not different from the NK rats. Histopathological examination of the OVDE group kidney showed protein deposition in the capsular of Bowman’s capsule and proximal tubules, atrophy of the proximal tubules, and necrosis, respectively. It is concluded that the combination of calcitriol with ethinyl ethyl estradiol in ovariectomized rats increased urinary Ca excretion and increased the risk of urolithiasis. ABSTRAK Tingginya ekskresi kalsium (Ca dalam urin dapat menjadi pemicu terbentuknya urolit. Estrogen dan calcitriol menurunkan ekskresi Ca urin. Penelitian ini dilakukan bertujuan untuk mengkaji kombinasi calcitriol dan ethynil ethyl estradiol terhadap ekskresi Ca dalam urin

  11. A dose-finding, cross-over study to evaluate the effect of a Nestorone®/Estradiol transdermal gel delivery on ovulation suppression in normal ovulating women.

    Science.gov (United States)

    Brache, Vivian; Merkatz, Ruth; Kumar, Narender; Jesam, Cristian; Sussman, Heather; Hoskin, Elena; Roberts, Kevin; Alami, Mohcine; Taylor, Deshawn; Jorge, Aidelis; Croxatto, Horacio; Lorange, Ellen; Mishell, Daniel R; Sitruk-Ware, Regine

    2015-10-01

    This study aims to determine the lowest effective of three Nestorone (NES)/estradiol (E2) transdermal gel doses to ensure ovulation suppression in 90-95% of cycles. This was a randomized, open-label, three-treatment-period cross-over study to evaluate the effects of NES/E2 transdermal gel on ovulation inhibition, suppression of follicular growth and pharmacokinetic parameters. The doses were low (1.5 mg NES/0.5 mg E2), medium (3.0 mg NES/1.0 mg E2) and high (4.5 mg NES/1.5 mg E2). Participants applied gel daily to a fixed area on the abdomen for 21 consecutive days. They were interviewed regarding their experiences using the gel. Eighteen participants were randomized; 16 completed the study. Median NES C(max) values for low, medium and high dose groups at day 21 were 318.6 pmol/L, 783.0 pmol/L and 1063.8 pmol/L, respectively. Median maximum follicular diameter was higher with the lowest dose with 16.2 mm versus 10.0 and 10.4 mm with the medium and high doses, respectively. Among adherent participants, ovulation was inhibited in all dose groups, except for one participant in the medium dose (6.7%) that had luteal activity and an ultrasound image suggestive of a luteinized unruptured follicle. There were few reports of unscheduled bleeding, with more episodes reported for the lower dose. Adverse events were mild, and no skin irritation was reported from gel application. While all three doses blocked ovulation effectively and were evaluated as safe and acceptable, the medium dose was considered the lowest effective dose based on a more adequate suppression of follicular development. Further development of this novel contraceptive delivering NES and E2 is warranted and has potential for improved safety compared to ethinyl-estradiol-based methods. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Naturally-occurring estradiol-17β-fatty acid esters, but not estradiol-17β, preferentially induce mammary tumorigenesis in female rats: Implications for an important role in human breast cancer

    International Nuclear Information System (INIS)

    Mills, Laura H.; Yu Jina; Xu Xiaomeng; Lee, Anthony J.; Zhu Baoting

    2008-01-01

    Because mammary glands are surrounded by adipose tissues, we hypothesize that the ultra-lipophilic endogenous estrogen-17β-fatty acid esters may have preferential hormonal and carcinogenic effects in mammary tissues compared to other target organs (such as the uterus and pituitary). This hypothesis is tested in the present study. We found that all 46 rats implanted with an estradiol-17β pellet developed large pituitary tumors (average weight = 251 ±103 mg) and had to be terminated early, but only 48% of them developed mammary tumors. In addition, approximately one-fourth of them developed a huge uterus. In the 26 animals implanted with a mixture containing estradiol-17β-stearate and estradiol-17β-palmitate (two representative estradiol-17β-fatty acid esters) or in the 29 animals implanted with estradiol-17β-stearate alone (in the same molar dose as estradiol-17β), 73% and 79%, respectively, of them developed mammary tumors, whereas only 3 or 2 animals, respectively, had to be terminated early due to the presence of a large pituitary tumor. Both tumorous and normal mammary tissues contained much higher levels of estrogen esterase than other tissues, which catalyzes the releases of bioactive estrogens from their fatty acid esters. In conclusion, while estradiol-17β is much stronger in inducing pituitary tumor (100% incidence) than mammary tumor, estradiol-17β-fatty acid esters have a higher efficacy than estradiol-17β in inducing mammary tumor and yet it only has little ability to induce uterine out-growth and pituitary tumorigenesis. This study establishes the endogenous estrogen-17β-fatty acid esters as preferential inducers of mammary tumorigenesis

  13. Interactions between androgens, FSH, anti-Müllerian hormone and estradiol during folliculogenesis in the human normal and polycystic ovary.

    Science.gov (United States)

    Dewailly, Didier; Robin, Geoffroy; Peigne, Maëliss; Decanter, Christine; Pigny, Pascal; Catteau-Jonard, Sophie

    2016-11-01

    Androgens, FSH, anti-Müllerian hormone (AMH) and estradiol (E2) are essential in human ovarian folliculogenesis. However, the interactions between these four players is not fully understood. The purpose of this review is to highlight the chronological sequence of the appearance and function of androgens, FSH, AMH and E2 and to discuss controversies in the relationship between FSH and AMH. A better understanding of this interaction could supplement our current knowledge about the pathophysiology of the polycystic ovary syndrome (PCOS). A literature review was performed using the following search terms: androgens, FSH, FSH receptor, anti-Mullerian hormone, AMHRII, estradiol, follicle, ovary, PCOS, aromatase, granulosa cell, oocyte. The time period searched was 1980-2015 and the databases interrogated were PubMed and Web of Science. During the pre-antral ('gonadotropin-independent') follicle growth, FSH is already active and promotes follicle growth in synergy with theca cell-derived androgens. Conversely, AMH is inhibitory by counteracting FSH. We challenge the hypothesis that AMH is regulated by androgens and propose rather an indirect effect through an androgen-dependent amplification of FSH action on granulosa cells (GCs) from small growing follicles. This hypothesis implies that FSH stimulates AMH expression. During the antral ('gonadotropin-dependent') follicle growth, E2 production results from FSH-dependent activation of aromatase. Conversely, AMH is inhibitory but the decline of its expression, amplified by E2, allows full expression of aromatase, characteristic of the large antral follicles. We propose a theoretical scheme made up of two triangles that follow each other chronologically. In PCOS, pre-antral follicle growth is excessive (triangle 1) because of intrinsic androgen excess that renders GCs hypersensitive to FSH, with consequently excessive AMH expression. Antral follicle growth and differentiation are disturbed (triangle 2) because of the

  14. Estradiol-induced promotion of hepatocarcinogenesis in medaka: Relationship of foci of cellular alteration to neoplasia

    Energy Technology Data Exchange (ETDEWEB)

    Cooke, J.B.; Hinton, D.E. [Univ. of California, Davis, CA (United States)

    1995-12-31

    In some laboratory and field studies, female fish have higher prevalences of liver tumors than do males. The authors hypothesize gender and site-specific differences in prevalence are due to variable exposures of previously initiated fish to tumor modulating compounds. Estradiol, a growth promoter, increases incidences of hepatic tumors in carcinogen-treated rainbow trout and medaka (Oryzias latipes). Estradiol also increases incidences of hepatic foci of cellular alteration (FCA) in medaka. FCA are found in subadults of tumor-bearing feral populations. Lack of knowledge about the relationship of various phenotypes of FCA to eventual tumors, however, has prevented use of FCA as a biomarker. The authors examined fate and growth of liver FCA using a 2-step, initiation-promotion protocol. Three week old medaka were exposed to 200 ppm diethylnitrosamine (DEN) for 24 hr. and then fed 0.1 ppm 17-{beta}-estradiol (E2) continuously through sampling at weeks 4--26. Percent volume of FCA and morphometric characteristics of normal and focal hepatocytes, including numerical density and average hepatocyte volume were quantified using computer-assisted stereology. E2 increased percentage of liver occupied by DEN-initiated amphophilic, basophilic and eosinophilic FCA in both sexes. Focal parameters of young, DEN-initiated and estradiol-treated medaka were not reached until much later in fish given only DEN. Non-focal hepatocytes in estradiol-treated medaka were smaller and more numerous than in DEN-only counterparts. Morphometric analysis is quantitatively tracking the fate of specific phenotypes of FCA to determine their role in progression to cancer.

  15. Treatment of labial adhesion with topical estrogen and correlation with serum estradiol level

    Directory of Open Access Journals (Sweden)

    Safaian B

    2013-05-01

    Full Text Available Background: Serum estradiol level is a controversial prognostic factor in the outcome of labial adhesion. The aim of this study was to evaluate serum estradiol levels and topical estrogen response in patients with labial adhesion.Methods: A prospective interventional study was conducted among girls with labial adhesion that referred to Pediatrics clinic in Taleghani University Hospital, Gorgan city, Iran in 2011. One hundred patients entered the study. The diagnosis was conducted by clinical examination of vestibule area. Inclusion criteria were, three months to eight years old prepuberty girls, no ambiguous genitalia, lack of vulvovaginitis symptoms, labial adhesion more than twenty five percent, no history of previous topical estrogen treatment since two weeks ago and previous incomplete treatment. The patients who did not use proper amount and duration of drug and also with adverse drug reactions during treatment period were excluded from the study. Results: The maximum frequency of labial adhesion was in the group of less than one year old. The minimum frequency of labial adhesion was in the 7-8 years old group. Eighty six patients had complete or partial remission. No evidence of an improvement was observed in fourteen children. Severity of adhesions did not worsen in our patients. Serum estradiol levels were lower in patients who had a positive response to treatment. There were significant differences in serum estradiol levels between full or relative improvement with no improvement groups (P=0.044.Conclusion: Findings of this study showed that the labial adhesion patients with low serum estradiol level had better treatment response after using topical estrogen.

  16. Effects of short-term administration of estradiol on reperfusion arrhythmias in rats of different ages

    International Nuclear Information System (INIS)

    Savergnini, S.Q.; Reis, A.M.; Santos, R.A.S.; Santos, P.E.B.; Ferreira, A.J.; Almeida, A.P.

    2012-01-01

    Little is known about age-related differences in short-term effects of estradiol on ischemia-reperfusion (I/R) insults. The present study was designed to evaluate the effects of short-term treatment with estradiol on reperfusion arrhythmias in isolated hearts of 6-7-week-old and 12-14-month-old female rats. Wistar rats were sham-operated, ovariectomized and treated with vehicle or ovariectomized and treated with 17β-estradiol (E 2 ; 5 µg·100 g −1 ·day −1 ) for 4 days. Hearts were perfused by the Langendorff technique. Reperfusion arrhythmias, i.e., ventricular tachycardia and/or ventricular fibrillation, were induced by 15 min of left coronary artery ligation and 30 min of reperfusion. The duration and incidence of I/R arrhythmias were significantly higher in young rats compared to middle-aged rats (arrhythmia severity index: 9.4 ± 1.0 vs 3.0 ± 0.3 arbitrary units, respectively, P < 0.05). In addition, middle-aged rats showed lower heart rate, systolic tension and coronary flow. Four-day E 2 treatment caused an increase in uterine weight. Although E 2 administration had no significant effect on the duration of I/R arrhythmias in middle-aged rats, it induced a marked reduction in the rhythm disturbances of young rats accompanied by a decrease in heart rate of isolated hearts. Also, this reduction was associated with an increase in QT interval. No significant changes were observed in the QT interval of middle-aged E 2 -treated rats. These data demonstrate that short-term estradiol treatment protects against I/R arrhythmias in hearts of young female rats. The anti-arrhythmogenic effect of estradiol might be related to a lengthening of the QT interval

  17. Effect of Estradiol Prescribed during Luteal Phase of Art Cycles and Pregnancy Outcome

    Directory of Open Access Journals (Sweden)

    M Karimzadeh

    2007-01-01

    Full Text Available Introduction: Implantation is one of the most important steps in ART cycles and it depends upon embryo and endometrial reception. Different protocols have been suggested for getting better endometrium. It seems estrogen increases the endometrial reception and pregnancy rate by inducing changes in the hormonal status. The aim of this study was to evaluate the effect of estradiol(E2 on luteal phase support and pregnancy rate in ART cycles Methods: This prospective randomized study was done in Yazd at the IVF center from March until December, 2002. 68 patients who had undergone IVF or ICSI were enrolled in the study. Exclusion criteria was age>40, endometriosis and ovarian hyper stimulation syndrome. Induction ovulation protocol was long suppression with GnRH analogues.After embryo transfer, patients were divided in two groups randomly. Both groups received 100mg progesterone IM daily from the transfer day. Estradiol valerate 2 mg/day was added from the 7th transfer day to progesterone in Group I and continued if the BhCG became positive. Abortion and malformations were measured in all patients. Data analyzed with SPSS 11.0 and P value <0.05 considered statistically significant. Results: Pregnancy rate in the 34 patients of estradiol group (group I was 26.5%which was significantly higher than 11.8 %( 4 cases in the other group (Pvalue=0.034. Abortion rate was higher in estradiol group (3 cases, but there was no abortion in the progesterone group(P=0.119. 2 cases of major fetal malformations were observed in E2 supplementation group (P=0.246 . Conclusions: E2 suplementation to progesterone in the luteal phase of ART cycles, especially in the long GnRH analogues causes higher endometrial receptivity and pregnancy rate.

  18. A study on the synthesis, labeling and its biodistribution of estradiol derivatives

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sang Wook; Yang, Seung Dae; Suh, Yong Sup [College of Medicine, Dongguk Univ., Seoul (Korea, Republic of)] [and others

    2000-10-01

    Due to the heterogeneous receptor distribution and changes of receptor status over time, the biochemical measurement of estrogen receptor status of biopsy specimens is not sufficient to diagnose breast cancer. As a result, I-123 labeled estradiols have been applied for the diagnosis. The purpose of this study was to develop a suitable radioligand for imaging estrogen receptor-positive human breast tumors. Among the various estradiol derivatives, 17{alpha}-[{sup 123}I]iodovinyl estradiol ([{sup 123}]IVE) has been prepared from 17{alpha}-ethynyl estradiol. Labeling of E-17{alpha}-[{sup 123}I]iodovinyl estradiol ([{sup 123}]IVE] was carried out using peracetic acid with [{sup 123}I]Nal and Z-[{sup 123}I]IVE labelling was archived using chloamine T/HCL solution with [{sup 123}I]Nal. Labeling yield was determined by silica thin-layer chromatography (TLC) and radiochemical purity was measured by high performance liquid chromatography (HPLC). The biodistribution of E-[{sup 123}I]IVE was measured in immature female rats at 60 min, 120 min and 300 min after injection. The labeling yield of two isomers was 92% and 94% (E-[{sup 123}I]IVE and Z-[{sup 123}I]IVE, respectively). The radiochemical purity was more than 98% after purification. The highest uptake was observed at 120 min in uterus (3.11% ID/g for E-[{sup 123}I]IVE. These results suggest the possibility of using E-[{sup 123}I]IVE as an imaging agent for the evaluation of the presence of estrogen receptor in patients with breast cancer.