RESUMO
OBJECTIVES: Most human in-vivo placental imaging techniques are unable to distinguish and characterize various placental compartments, such as the intervillous space (IVS), placental vessels (PV) and placental tissue (PT), limiting their specificity. We describe a method that employs T2* and diffusion-weighted magnetic resonance imaging (MRI) data to differentiate automatically placental compartments, quantify their oxygenation properties and identify placental lesions (PL) in vivo. We also investigate the association between placental oxygenation patterns and fetal brain oxygenation. METHODS: This was a prospective study conducted between 2018 and 2021 in which dual-contrast clinical MRI data (T2* and diffusion-weighted MRI) were acquired from patients between 20 and 38 weeks' gestation. We trained a fuzzy clustering method to analyze T2* and diffusion-weighted MRI data and assign placental voxels to one of four clusters, based on their distinct imaging domain features. The new method divided automatically the placenta into IVS, PV, PT and PL compartments and characterized their oxygenation changes throughout pregnancy. RESULTS: A total of 27 patients were recruited, of whom five developed pregnancy complications. Total placental oxygenation level and T2* did not demonstrate a statistically significant temporal correlation with gestational age (GA) (R2 = 0.060, P = 0.27). In contrast, the oxygenation level reflected by T2* values in the placental IVS (R2 = 0.51, P = 0.0002) and PV (R2 = 0.76, P = 1.1 × 10-7 ) decreased significantly with advancing GA. Oxygenation levels in the PT did not show any temporal change during pregnancy (R2 = 0.00044, P = 0.93). A strong spatial-dependent correlation between PV oxygenation level and GA was observed. The strongest negative correlation between PV oxygenation and GA (R2 = 0.73, P = 4.5 × 10-7 ) was found at the fetal-vessel-dominated region close to the chorionic plate. The location and extent of the placental abnormality were automatically delineated and quantified in the five women with clinically confirmed placental pathology. Compared to the averaged total placental oxygenation, placental IVS oxygenation level best reflected fetal brain oxygenation level during fetal development. CONCLUSION: Based on clinically feasible dual-MRI, our method enables accurate spatiotemporal quantification of placental compartment and fetal brain oxygenation across different GAs. This information should improve our knowledge of human placenta development and its relationship with normal and abnormal pregnancy. © 2022 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.
Assuntos
Doenças Placentárias , Complicações na Gravidez , Gravidez , Feminino , Humanos , Placenta/diagnóstico por imagem , Placenta/patologia , Estudos Prospectivos , Doenças Placentárias/diagnóstico por imagem , Doenças Placentárias/patologia , Imageamento por Ressonância Magnética/métodos , Placentação , Complicações na Gravidez/patologia , Encéfalo/diagnóstico por imagemRESUMO
BMS-986020, BMS-986234 and BMS-986278, are three lysophosphatidic acid receptor 1 (LPA1) antagonists that were or are being investigated for treatment of idiopathic pulmonary fibrosis (IPF). Hepatobiliary toxicity (elevated serum AST, ALT, and ALP, plasma bile acids [BAs], and cholecystitis) was observed in a Phase 2 clinical trial with BMS-986020, and development was discontinued. In dogs and rats, the species used for the pivotal toxicology studies, there was no evidence of hepatobiliary toxicity in the dog while findings in the rat were limited to increased plasma BAs levels (6.1× control), ALT (2.9×) and bilirubin (3.4×) with no histopathologic correlates. Since neither rats nor dogs predicted clinical toxicity, follow-up studies in cynomolgus monkeys revealed hepatobiliary toxicity that included increased ALT (2.0× control) and GLDH (4.9×), bile duct hyperplasia, cholangitis, cholestasis, and cholecystitis at clinically relevant BMS-986020 exposures with no changes in plasma or liver BAs. This confirmed monkey as a relevant species for identifying hepatobiliary toxicity with BMS-986020. In order to assess whether the toxicity was compound-specific or related to LPA1 antagonism, two structurally distinct LPA1 antagonists (BMS-986234 and BMS-986278), were evaluated in rat and monkey. There were no clinical or anatomic pathology changes indicative of hepatobiliary toxicity. Mixed effects on plasma BAs in both rat and monkey has made this biomarker not a useful predictor of the hepatobiliary toxicity. In conclusion, the nonclinical data indicate the hepatobiliary toxicity observed clinically and in monkeys administered BMS-986020 is compound specific and not mediated via antagonism of LPA1.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doenças do Sistema Digestório/induzido quimicamente , Fígado/efeitos dos fármacos , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Animais , Ácidos e Sais Biliares/sangue , Bilirrubina/sangue , Doença Hepática Induzida por Substâncias e Drogas/sangue , Doenças do Sistema Digestório/sangue , Doenças do Sistema Digestório/metabolismo , Cães , Feminino , Haplorrinos , Fígado/metabolismo , Hepatopatias/sangue , Hepatopatias/metabolismo , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Syncytial nuclear aggregates (SNAs), clusters of nuclei in the syncytiotrophoblast of the human placenta, are increased as gestation advances and in pregnancy pathologies. The origins of increased SNAs are unclear; however, a better appreciation of the mechanism may give insight into placental ageing and factors underpinning dysfunction. We developed three models to investigate whether SNA formation results from a dynamic process of nuclear movement and to generate alternative hypotheses. SNA count and size were measured in placental explants cultured over 16 days and particles released into culture medium were quantified. Primary trophoblasts were cultured for 6 days. Explants and trophoblasts were cultured with and without cytoskeletal inhibitors. An in silico model was developed to examine the effects of modulating nuclear behaviour on clustering. In explants, neither median SNA number (108 SNA/mm(2) villous area) nor size (283 µm(2)) changed over time. Subcellular particles from conditioned culture medium showed a wide range of sizes that overlapped with those of SNAs. Nuclei in primary trophoblasts did not change position relative to other nuclei; apparent movement was associated with positional changes of the syncytial cell membrane. In both models, SNAs and nuclear clusters were stable despite pharmacological disruption of cytoskeletal activity. In silico, increased nuclear movement, adhesiveness and sites of cytotrophoblast fusion were related to nuclear clustering. The prominence of SNAs in pregnancy disorders may not result from an active process involving cytoskeleton-mediated rearrangement of syncytial nuclei. Further insights into the mechanism(s) of SNA formation will aid understanding of their increased presence in pregnancy pathologies.
Assuntos
Membrana Celular/ultraestrutura , Núcleo Celular/ultraestrutura , Citoesqueleto/ultraestrutura , Placenta/ultraestrutura , Trofoblastos/ultraestrutura , Feminino , Imunofluorescência , Humanos , Gravidez , Imagem com Lapso de TempoRESUMO
Workshops are an important part of the IFPA annual meeting. At the IFPA meeting 2008 diverse topics were discussed in 12 themed workshops. Topics covered included: immunology of placentation; galectins and trophoblast invasion; signaling in implantation and invasion; markers to identify trophoblast subpopulations; placental pathology; placental toxicology; stereology; placental transport of fatty acids; placental mesenchymal stem cells; comparative placentation; trophoblast and neoplasia; trophoblast differentiation. This report is a summary of the various topics covered.
Assuntos
Placenta/fisiologia , Placentação/imunologia , Trofoblastos/fisiologia , Animais , Feminino , Humanos , Placenta/imunologia , Doenças Placentárias/imunologia , GravidezRESUMO
Electron microscope autoradiography has been used to study protein synthesis in syncytial and cellular trophoblast of term human placental villi incubated in vitro with tritiated leucine ([3H]leu). Autoradiographs were analyzed using the hypothetical grain analysis of Blackett and Parry (1973. J. Cell Biol. 57:9-15). The results of this study demonstrated that both cellular and syncytial trophoblast have marked capacities for protein synthesis. Cellular trophoblast synthesized protein in both its rough endoplasmic reticulum (RER) and its ground plasm which contained abundant free ribosomes. The vast majority of 3H-proteins remained within the cell, with some of the proteins synthesized ultimately appearing in the nucleus. A small percentage of grains was ultimately associated with the trophoblast basement membrane. In syncytial trophoblast, the RER was the dominant site for protein synthesis. The autoradiographic data suggested that, as in the cellular trophoblast, the vast majority of 3H-proteins synthesized by the syncytial trophoblast remained within the syncytial trophoblast throughout the incubation period. The major portion of [3H]leu-labeling present in the syncytial trophoblast of villi incubated the longest times (4 h+) remained in association with the RER. Labeled proteins did not become concentrated in syncytial trophoblast Golgi apparatus, vesicles, or granules. In contrast to cellular trophoblast, the nuclei in the syncytium did not contain 3H-proteins at any time-point studied.
Assuntos
Leucina/metabolismo , Organelas/metabolismo , Placenta/citologia , Biossíntese de Proteínas , Trofoblastos/metabolismo , Trofoblastos/ultraestrutura , Autorradiografia , Membrana Basal/metabolismo , Membrana Basal/ultraestrutura , Células Cultivadas , Retículo Endoplasmático Rugoso/metabolismo , Retículo Endoplasmático Rugoso/ultraestrutura , Feminino , Humanos , Microscopia Eletrônica , Organelas/ultraestrutura , Placenta/metabolismo , Gravidez , Ribossomos/metabolismo , Ribossomos/ultraestrutura , TrítioRESUMO
Electron microscope autoradiography was used to study glycoprotein synthesis in cellular trophoblast (cytotrophoblast) and syncytial trophoblast of term human placental villi incubated in vitro with D-[1-3H]galactose ([3H]gal). Autoradiographs were analyzed using the hypothetical grain analysis of Blackett and Parry (1973. J. Cell Biol. 57:9-15). The results of this study indicated that [3H]gal incorporation into term placental villi was predominantly localized to cytotrophoblast. Utilization of [3H]gal by term syncytial trophoblast was extremely low and yielded too few grains for a quantitative grain analysis. This result is in striking contrast to that found in the preceding study of [3H]leucine incorporation (Nelson, D. M., A. C. Enders, and B. F. King. 1978). Within cytotrophoblast, the rough endoplasmic reticulum incorporated the most [3H]gal into glycoprotein. The Golgi apparatus was another site of [3H]gal incorporation. The vast majority of the [3H]gal incorporated into cytotrophoblast during the pulse incubation remained intracellular through the duration of the experiment. There was little autoradiographic evidence for secretion of tritiated macromolecules. Cytotrophoblast incubated for the longest time period studied (4 h+) showed a substantial concentration of tritiated macromolecules in the Golgi complex and in the ground plasm but not in the rough endoplasmic reticulum.
Assuntos
Galactose/metabolismo , Glicoproteínas/biossíntese , Organelas/metabolismo , Organelas/ultraestrutura , Placenta/citologia , Trofoblastos/citologia , Trofoblastos/metabolismo , Autorradiografia , Retículo Endoplasmático Rugoso/metabolismo , Retículo Endoplasmático Rugoso/ultraestrutura , Feminino , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Humanos , Microscopia Eletrônica , Placenta/metabolismo , Gravidez , Trítio , Trofoblastos/ultraestruturaRESUMO
Measurements of chlorophyll, particulate carbon, and biogenic silica concentrations near a receding ice edge off the coast of Victoria Land, Antarctica, indicated the presence of a dense phytoplankton bloom. The bloom extended 250 kilometers from the ice edge and was restricted to waters where the melting of ice had resulted in reduced salinity. The region involved was one of enhanced vertical stability, which may have favored phytoplankton growth, accumulation, or both. Epontic algae released from melting ice may have served as an inoculum for the bloom. Ratios of organic carbon to chlorophyll and biogenic silica to carbon were unusually high, resulting in high biogenic silica concentrations despite only moderately high chlorophyll levels.
RESUMO
The net inputs of silicic acid (dissolved silica) to the world ocean have been revised to 6.1 +/- 2.0 teramoles of silicon per year (1 teramole = 10(12) moles). The major contribution (about 80 percent) comes from rivers, whose world average silicic acid concentration is 150 micromolar. These inputs are reasonably balanced by the net ouputs of biogenic silica of 7.1 +/- 1.8 teramoles of silicon per year in modern marine sediments. The gross production of biogenic silica (the transformation of dissolved silicate to particulate skeletal material) in surface waters was estimated to be 240 +/- 40 teramoles of silicon per year, and the preservation ratio (opal accumulation in sediment/gross production in surface waters) averages 3 percent. In the world ocean the residence time of silicon, relative to total biological uptake in surface waters, is about 400 years.
RESUMO
The complement system plays an important role in normal human pregnancy. Uncontrolled activation of this system has been associated with many disease states. We tested the hypothesis that the C5b-9 membrane attack complex (MAC) localizes to sites of villous injury and modulates trophoblast function. Placental sections from pregnancies with no complications, intrauterine growth restriction, or preeclampsia were immunostained and the surface density for MAC and fibrin was determined by morphometric analysis. Primary cytotrophoblasts from term placentas were cultured in a FiO(2) of <1%, 8% and 20% with 10% human serum containing active MAC or heat-inactivated control serum. Immunofluorescent MAC binding to trophoblast was quantified, and the neoepitopes formed in cytokeratin 18 filaments and poly-ADP-ribose polymerase during apoptosis were used to measure cell death. Trophoblast differentiation was assessed by HCG secretion, formation of syncytia, and expression of syncytin. MAC localized to fibrin deposits in normal placentas, and especially in placentas from IUGR and preeclampsia. MAC binding to cytotrophoblasts was inversely proportional to FiO(2) and enhanced apoptosis. MAC increased markers of differentiation in cultures at 72h (medium HCG, syncytia and syncytin expression). Our findings demonstrate that MAC associates with fibrin deposits at sites of villous injury in vivo. Hypoxia also enhances MAC deposition in cultured trophoblasts and MAC alters trophoblast function in a phenotype specific manner.
Assuntos
Vilosidades Coriônicas/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Doenças Placentárias/metabolismo , Trofoblastos/metabolismo , Apoptose/fisiologia , Vilosidades Coriônicas/patologia , Complexo de Ataque à Membrana do Sistema Complemento/fisiologia , Feminino , Retardo do Crescimento Fetal/fisiopatologia , Fibrina/metabolismo , Humanos , Doenças Placentárias/patologia , Pré-Eclâmpsia/fisiopatologia , GravidezRESUMO
Hypoxic injury hinders placental differentiation and alters trophoblast gene expression. We tested the hypothesis that the expression of follistatin-like 3 (FSTL3), a member of the follistatin family of proteins, is modulated by hypoxia in primary human trophoblast (PHT). Using immunofluorescence of human term placental villi we detected the expression of FSTL3 protein in placental villi, primarily in trophoblasts. We verified this finding in cultured term PHT cells. Basal expression of FSTL3 transcript in cultured PHT cells, determined using quantitative PCR, was stable over the culture period. Importantly, when compared to culture in FiO(2)=20% or FiO(2)=8%, PHT cells cultured in FiO(2) <1% exhibited a 4-6 fold increase in FSTL3 mRNA expression as early as 4h in hypoxia. Whereas cellular FSTL3 protein was unchanged in hypoxia, we found that hypoxia increased the level of FSTL3 in the medium. Lastly, the exposure of PHT cells to either the hypoxia-mimetic cobalt chloride or the proline hydroxylase inhibitor dimethyloxaloylglycine upregulated the expression of FSTL3 transcript. Our data indicate that hypoxia enhances the expression of FSTL3 and its release from PHT cells. Our finding that hypoxia-mimetic agents enhance FSTL3 expression implicates HIF1alpha in this process.
Assuntos
Proteínas Relacionadas à Folistatina/metabolismo , Hipóxia/metabolismo , Trofoblastos/metabolismo , Feminino , Proteínas Relacionadas à Folistatina/análise , Proteínas Relacionadas à Folistatina/genética , Humanos , Hipóxia/genética , Gravidez , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Nascimento a Termo , Trofoblastos/químicaRESUMO
We investigated the mechanisms of alphafetoprotein (AFP) transfer across the human placenta by correlating measurements of AFP transfer with cytochemical localization of AFP. Placental cotyledons were dually perfused in vitro with either the fetal or maternal perfusate containing umbilical cord plasma as a source of AFP. Steady state AFP clearance, corrected for release of endogenous AFP, was 0.973 +/- 0.292 microliter/min per gram in the fetal to maternal direction (n = 10), significantly higher (P < 0.02) than that in the maternal to fetal direction (n = 5; 0.022 +/- 0.013 microliter/min per gram). Clearance of a similarly sized protein, horseradish peroxidase was also asymmetric but clearance of the small tracer creatinine was not. Using a monoclonal antibody, we localized AFP to fibrinoid deposits in regions of villi with discontinuities of the syncytiotrophoblast, to cytotrophoblast cells in these deposits, to syncytiotrophoblast on some villi, and to trophoblast cells in the decidua. We conclude that AFP transfer in the placenta is asymmetric and that there are two available pathways for AFP transfer: (a) from the fetal circulation into the villous core and across fibrinoid deposits at discontinuities in the villous syncytiotrophoblast to enter the maternal circulation; and (b) AFP present in the decidua could enter vessels that traverse the basal plate.
Assuntos
Troca Materno-Fetal , Placenta/metabolismo , alfa-Fetoproteínas/metabolismo , Transporte Biológico , Feminino , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Perfusão , Gravidez , alfa-Fetoproteínas/análiseRESUMO
Hypoxia influences gene expression in placental trophoblasts. We sought to examine the effect of hypoxia on trophoblast expression of human PHLDA2 (also termed IPL, TSSC3 or BWR-1C), a product of an imprinted gene on human chromosome 11p15.5 whose murine ortholog plays a pivotal role in placental development. We initially confirmed that PHLDA2 was expressed in term placental villi, primarily in the trophoblast layer. Using quantitative PCR we found that the expression of PHLDA2 gradually declined during differentiation of primary term human trophoblasts. A similar expression pattern was seen for p57(Kip2) and IGF-II, both products of imprinted genes on chromosome 11p15.5. Exposure of trophoblasts to hypoxia in vitro (O(2)Assuntos
Inibidor de Quinase Dependente de Ciclina p57/metabolismo
, Hipóxia/metabolismo
, Fator de Crescimento Insulin-Like II/metabolismo
, Proteínas Nucleares/metabolismo
, Trofoblastos/metabolismo
, Células Cultivadas
, Humanos
RESUMO
Substrates of cyclin-cdk2 kinases contain two distinct primary sequence motifs: a cyclin-binding RXL motif and one or more phosphoacceptor sites (consensus S/TPXK/R or S/TP). To identify novel cyclin-cdk2 substrates, we searched the database for proteins containing both of these motifs. One such protein is human HIRA, the homologue of two cell cycle-regulated repressors of histone gene expression in Saccharomyces cerevisiae, Hir1p and Hir2p. Here we demonstrate that human HIRA is an in vivo substrate of a cyclin-cdk2 kinase. First, HIRA bound to and was phosphorylated by cyclin A- and E-cdk2 in vitro in an RXL-dependent manner. Second, HIRA was phosphorylated in vivo on two consensus cyclin-cdk2 phosphoacceptor sites and at least one of these, threonine 555, was phosphorylated by cyclin A-cdk2 in vitro. Third, phosphorylation of HIRA in vivo was blocked by cyclin-cdk2 inhibitor p21(cip1). Fourth, HIRA became phosphorylated on threonine 555 in S phase when cyclin-cdk2 kinases are active. Fifth, HIRA was localized preferentially to the nucleus, where active cyclin A- and E-cdk2 are located. Finally, ectopic expression of HIRA in cells caused arrest in S phase and this is consistent with the notion that it is a cyclin-cdk2 substrate that has a role in control of the cell cycle.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Ciclo Celular , Ciclina A/metabolismo , Ciclina E/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição/química , Fatores de Transcrição/fisiologia , Sequência de Aminoácidos , Western Blotting , Ciclo Celular , Linhagem Celular , Núcleo Celular/metabolismo , Separação Celular , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Citometria de Fluxo , Glutationa Transferase/metabolismo , Chaperonas de Histonas , Humanos , Espectrometria de Massas , Microscopia de Fluorescência , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Peptídeos/química , Fosforilação , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/química , Fase S , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Treonina/química , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
Forests cover 30% of the terrestrial Earth surface and are a major component of the global carbon (C) cycle. Humans have doubled the amount of global reactive nitrogen (N), increasing deposition of N onto forests worldwide. However, other global changes-especially climate change and elevated atmospheric carbon dioxide concentrations-are increasing demand for N, the element limiting primary productivity in temperate forests, which could be reducing N availability. To determine the long-term, integrated effects of global changes on forest N cycling, we measured stable N isotopes in wood, a proxy for N supply relative to demand, on large spatial and temporal scales across the continental U.S.A. Here, we show that forest N availability has generally declined across much of the U.S. since at least 1850 C.E. with cool, wet forests demonstrating the greatest declines. Across sites, recent trajectories of N availability were independent of recent atmospheric N deposition rates, implying a minor role for modern N deposition on the trajectory of N status of North American forests. Our results demonstrate that current trends of global changes are likely to be consistent with forest oligotrophication into the foreseeable future, further constraining forest C fixation and potentially storage.
Assuntos
Florestas , Nitrogênio/análise , Madeira/química , Clima , Humanos , Ciclo do Nitrogênio , Análise Espaço-Temporal , Estados UnidosRESUMO
We tested the hypothesis that the expression levels of p53 and the pro-apoptotic mediators from the Bcl-2 family are higher in cytotrophoblasts, when compared to cultures with abundant syncytiotrophoblasts. Cytotrophoblasts isolated from normal term human placentas were cultured in Dulbecco's Modified Eagle medium (DMEM) for 24 h, when the cytotrophoblast phenotype predominates, in DMEM for 72 h, when the syncytiotrophoblast phenotype predominates, or in Ham's-Waymouth medium or DMEM with 1.5% dimethylsulfoxide, each of which maintains the cytotrophoblast phenotype through 72 h of culture. Apoptosis was assessed by detection of cleavage products of poly-ADP-ribose polymerase, by expression of cleaved cytokeratin 18 intermediate filaments, and by assessment of caspase-3 activity. Independent of time in culture, cytotrophoblasts showed higher levels of apoptosis compared to syncytiotrophoblasts. Cytotrophoblasts also expressed a 2-fold higher level of p53, a 2-fold lower level of 60 kDa Mdm-2 protein, a 2-fold higher level of Bak, but no differences in the expression of 90 kDa Mdm-2, Bcl-2, Bcl-X(L), Mcl-1, Bax, Bad, and Bad phosphorylated at the serine(112), serine(136), or serine(155) sites, compared to the syncytiotrophoblasts. Using co-immunoprecipitation, we demonstrated a greater degree of Bak-p53 interaction in cytotrophoblasts than in syncytiotrophoblasts. We also detected Bak-Mcl-1 interaction that was no different between the two phenotypes. Among the proteins studied, enhanced p53 activity, differential Bak expression, and Bak-p53 interactions may contribute to the higher level of constitutive apoptosis in cultures of cytotrophoblasts compared to syncytiotrophoblasts.
Assuntos
Apoptose/fisiologia , Trofoblastos/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Células Cultivadas , Feminino , Humanos , Imunoprecipitação , Fenótipo , GravidezRESUMO
Major advances in placental biology have been realized as new technologies have been developed and existing methods have been refined in many areas of biological research. Classical anatomy and whole-organ physiology tools once used to analyze placental structure and function have been supplanted by more sophisticated techniques adapted from molecular biology, proteomics, and computational biology and bioinformatics. In addition, significant refinements in morphological study of the placenta and its constituent cell types have improved our ability to assess form and function in highly integrated manner. To offer an overview of modern technologies used by investigators to study the placenta, this workshop: Advanced techniques in placental biology, assembled experts who discussed fundamental principles and real time examples of four separate methodologies. Y. Sadovsky presented the principles of microRNA function as an endogenous mechanism of gene regulation. J. Robinson demonstrated the utility of correlative microscopy in which light-level and transmission electron microscopy are combined to provide cellular and subcellular views of placental cells. A. Croy provided a lecture on the use of microdissection techniques which are invaluable for isolating very small subsets of cell types for molecular analysis. Finally, G. Rice presented an overview methods on profiling of complex protein mixtures within tissue and/or fluid samples that, when refined, will offer databases that will underpin a systems approach to modern trophoblast biology.
Assuntos
MicroRNAs/fisiologia , Microdissecção/métodos , Micromanipulação/métodos , Microscopia Imunoeletrônica/métodos , Placenta/fisiologia , Análise Serial de Proteínas/métodos , Animais , Crioultramicrotomia/métodos , Feminino , Imunofluorescência , Regulação da Expressão Gênica , Humanos , Linfócitos/metabolismo , Micromanipulação/instrumentação , Útero/metabolismoRESUMO
Deficiency of either the mitogen-activated protein kinase p38 or the nuclear receptor PPARgamma results in disrupted vasculogenesis and abnormal development of the murine placenta. In addition, PPARgamma regulates differentiation of human trophoblasts. Here we tested the hypothesis that p38 plays an important role in the regulation of PPARgamma in primary human trophoblasts. We initially confirmed that cultured trophoblasts derived from normal term human placentas express p38 as well as its functional phosphorylated form. Whereas PPARgamma did not alter p38 expression, p38 inhibitors diminished the transcriptional activity of PPARgamma in primary trophoblasts. In addition, inhibition of p38 resulted in marked attenuation of PPARgamma-stimulated hCG production by cultured trophoblast. Our data support an effect of p38 on PPARgamma protein stability because p38 inhibition led to reduced expression of PPARgamma protein without a significant effect on PPARgamma mRNA, and this reduction was blocked by the protease inhibitor MG-132. Together, these data indicate that p38 regulates PPARgamma expression and activity in term human trophoblasts. Cross talk between p38 and PPARgamma signaling may play a role in modulating differentiation and function of the human placenta.
Assuntos
PPAR gama/metabolismo , Trofoblastos/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Células Cultivadas , Gonadotropina Coriônica/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Feminino , Humanos , Leupeptinas/farmacologia , PPAR gama/genética , Transcrição Gênica , Trofoblastos/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
These studies were designed to investigate the cytologic localization and topographic distribution of insulin receptors in human placental villi. Biochemical studies showed placental villi to specifically bind 125I-insulin. Radioautographic studies showed the specific binding to be localized to the surface of the syncytial trophoblast. Topographic distribution of insulin binding was determined with ferritin-insulin. Initial studies using ferritin-insulin containing some oligomers of ferritin revealed the insulin receptors to be specifically associated with the glycocalyx region of the surface membranes of microvilli. No insulin receptors were detectable in association with the intermicrovillous plasma membrane even though its glycocalyx is in direct continuity with the glycocalyx of microvilli. Monomeric ferritin-insulin showed the same nonuniform distribution of the insulin receptor, which suggests that there is not complete freedom of lateral mobility of the insulin receptors in the surface membrane of this tissue. The insulin receptors were found to occur as singletons or in groups of two or more. Incubations with monomeric ferritin-insulin at 4 degrees or with tissue prefixed with formaldehyde showed that the groups of insulin receptors were naturally occurring, i.e., they are present prior to and independent of insulin binding and thus not secondary to ligand-induced aggregation. The physiologic meaning of the nonuniform distribution and the groups of insulin receptors is unclear at present.
Assuntos
Membrana Celular/metabolismo , Microvilosidades/metabolismo , Placenta/metabolismo , Receptor de Insulina/metabolismo , Feminino , Humanos , Insulina/metabolismo , Microscopia Eletrônica , Microvilosidades/ultraestrutura , Placenta/ultraestrutura , GravidezRESUMO
Although prostaglandin E2 (PGE2) is known to inhibit glucose-induced insulin secretion, it is uncertain whether PGE2 actions on the beta-cell are direct, whether they are equipotent for both phases of hormone secretion, and whether the same mechanism of action prevails throughout. Study of the HIT cell, a clonal line of pancreatic beta-cells, provides answers to these questions because perifusion with glucose and 3-isobutyl-1-methylxanthine stimulates biphasic insulin secretion. Perifusion with PGE2 decreased both the first and second phases of glucose-induced insulin release to 47 +/- 4% of controls. Pretreatment with pertussis toxin partly prevented PGE2 inhibition to 80 +/- 4% of controls for first phase and 79 +/- 4% of controls for second phase. To evaluate whether the partial prevention of PGE2 inhibition seen with pertussis toxin pretreatment was caused by Gi heterotrimer association between the preincubation period and the end of perifusion, PGE2 actions were also examined during continuous treatment with pertussis toxin. Under these conditions, PGE2 inhibition of both phases was totally prevented. However, no difference was observed in membrane protein ADP ribosylation when cells were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after pretreatment or continuous treatment with pertussis toxin. Cyclic AMP (cAMP) accumulation was inhibited by PGE2 (from 3263 +/- 153 to 1549 +/- 158 fmol/10(6) cells) but less so after pretreatment with pertussis toxin (correlation between insulin release and cAMP accumulation during perifusion; n = 18, r = .85, P less than .001). Thus, PGE2 equally inhibits both phases of glucose-induced insulin secretion and cAMP generation through a pertussis toxin-sensitive G protein-mediated direct effect on the pancreatic beta-cell.
Assuntos
AMP Cíclico/metabolismo , Dinoprostona/farmacologia , Proteínas de Ligação ao GTP/fisiologia , Glucose/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Toxina Pertussis , Fatores de Virulência de Bordetella/farmacologia , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Linhagem Celular , Células Cultivadas , Células Clonais , Cricetinae , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacosRESUMO
Using oligonucleotide microarrays we recently identified a set of transcripts that were up-regulated in hypoxic human trophoblasts. To test the hypothesis that expression of hypoxia-related placental transcripts depends on sampling site we analyzed nine different sites from term human placentas (n=6), obtained after uncomplicated pregnancies. These sites spanned the placental center to the lateral border and the basal to the chorionic plate. Relative gene expression at each site, determined using quantitative PCR, was correlated with villous histology. The expression of vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF), the cytoskeleton proteins lamininA3 and alpha-tubulin, and the signal transduction protein Rad was enhanced in the subchorionic lateral border compared to medial basal site (1.6-2.9 fold, p<0.05). In contrast, the expression of NDRG1, adipophilin and human placental lactogen was unchanged. Enhanced villous maturation, syncytial knots and fibrin deposits were more frequent in the subchorionic placental lateral border, and correlated with up-regulation of hypoxia-related transcripts (p<0.05). The association between sample site and expression level was not observed in placentas with marginal cord insertion. The expression of hypoxia-related genes in the term human placenta is dependent on sampling site within the placental disk, likely reflecting local differences in villous perfusion.