RESUMO
Persistent breeding induced endometritis (PBIE) is a significant cause of infertility in mares. The development of a safe, universal, readily available therapeutic to manage PBIE and facilitate an optimal uterine environment for embryo development may improve pregnancy rates in susceptible mares. Mesenchymal stromal cells (MSCs) are being used increasingly as a therapeutic mediator for inflammatory conditions such as endometritis, and early gestational tissue provides a unique source of multipotent stem cells for creating MSCs. Extracellular vesicles (EVs) are mediators of cell communication produced by many different cell types. This study utilized embryo-derived mesenchymal stromal cells (EDMSCs) and their EVs as a potential therapeutic modality for PBIE in two groups: a) PBIE-susceptible mares challenged with pooled dead sperm (n=5); and b) client-owned mares diagnosed as susceptible to PBIE (n=37 mares and 40 estrous cycles). Mares pre-treated with intrauterine EDMSCs or their EVs resulted in a significant reduction in the accumulation of intrauterine fluid post-breeding. Nine of 19 (47 %) mares treated with EDMSCs prior to natural breeding and 13 of 20 (65 %) mares treated with EDMSC derived EVs were pregnant after the first cycle and 12 of 18 (67 %) mares treated with EDMSCs, and 15 of 19 (79 %) mares treated with EVs conceived by the end of the breeding season. These preliminary clinical studies are the first reports of the use of EDMSCs or their EVs as a potential intrauterine therapy for the management of PBIE susceptible mares.
Assuntos
Endometrite , Vesículas Extracelulares , Doenças dos Cavalos , Células-Tronco Mesenquimais , Animais , Cavalos , Feminino , Endometrite/veterinária , Endometrite/terapia , Células-Tronco Mesenquimais/metabolismo , Vesículas Extracelulares/transplante , Vesículas Extracelulares/metabolismo , Doenças dos Cavalos/terapia , Gravidez , Transplante de Células-Tronco Mesenquimais/métodos , Transplante de Células-Tronco Mesenquimais/veterinária , Cruzamento/métodos , Embrião de MamíferosRESUMO
INTRODUCTION: Primary trophoblast cultures obtained from term placentae are an important research tool. Term trophoblasts, while isolated as mononuclear cells, spontaneously fuse to form multinucleated syncytial clusters. Since term trophoblast cells do not replicate in vitro, contaminating cells can overgrow the culture limiting the lifespan of primary trophoblast cultures to about seven days. We aimed to develop a method that would allow the prolonged culture of term trophoblasts. METHODS: Trophoblasts were isolated from term placentae, following vaginal or cesarean section delivery, using either trypsin/DNase or dispase/DNase to digest the tissue. Purity of the trophoblasts was confirmed using flow cytometry prior to plating and during culture using immunocytochemistry. Cell death was examined with propidium iodide and trophoblast fusion monitored using PKH67 membrane stain. RESULTS: Digestion of term villous tissue with dispase/DNase resulted in the release of significantly more trophoblasts than digestion with trypsin/DNase (n = 8, p = 0.0051). Viability of the trophoblasts was unaffected by enzyme choice. The use of Advanced DMEM/F12 supplemented with 2% fetal bovine serum allowed culture of the trophoblasts with minimal cell death or contamination for 30 days. Despite prolonged culture over half of the trophoblasts remained mononuclear. DISCUSSION: We report a simple, optimized method to isolate and culture trophoblasts from term placentae for prolonged periods without substantial contamination with other cell types. Consistent with previous findings, trophoblasts cultured using our method were able to syncytialise, forming multi-nucleated syncytia. This extended growth time allows long term in vitro experimentation to further understand the nature of trophoblasts.
Assuntos
Técnicas de Cultura de Células , Separação Celular/métodos , Trofoblastos , Feminino , Humanos , GravidezRESUMO
BACKGROUND: Preeclampsia and intrauterine fetal growth restriction (IUGR) are two of the most common causes of maternal and perinatal morbidity and mortality. Current methods of predicting those at most risk of these conditions remain relatively poor, and in clinical practice past obstetric history remains the most commonly used tool. Aspirin and, in women at risk of preeclampsia only, calcium have been demonstrated to have a modest effect on risk reduction. Several observational studies and randomised trials suggest that low molecular weight heparin (LMWH) therapy may confer some benefit. METHODS/DESIGN: This is a multicentre open label randomised controlled trial to determine the effect of the LMWH, enoxaparin, on the prevention of recurrence of preeclampsia and/or IUGR in women at high risk due to their past obstetric history in addition to standard high risk care for all participants. INCLUSION CRITERIA: A singleton pregnancy >6+0 and <16+0 weeks gestation with most recent prior pregnancy with duration >12 weeks having; (1) preeclampsia delivered <36+0 weeks, (2) Small for gestational age (SGA) infant <10th customised birthweight centile delivered <36+0 weeks or, (3) SGA infant ≤3rd customised birthweight centile delivered at any gestation. Randomisation is stratified for maternal thrombophilia status and women are randomly assigned to 'standard high risk care' or 'standard high risk care' plus enoxaparin 40 mg from recruitment until 36+0 weeks or delivery, whichever occurs sooner. Standard high risk care includes the use of aspirin 100 mg daily and calcium 1000-1500 mg daily (unless only had previous SGA with no preeclampsia). The primary outcome is preeclampsia and/or SGA <5th customised birthweight centile. Analysis will be by intention to treat. DISCUSSION: The EPPI trial has more focussed and clinically relevant inclusion criteria than other randomised trials with a more restricted composite primary outcome. The inclusion of standard use of aspirin (and calcium) for all participants will help to ensure that any differences observed in outcome are likely to be related to enoxaparin use. These data will make a significant contribution to future meta-analyses and systematic reviews on the use of LMWH for the prevention of placental mediated conditions. TRIAL REGISTRATION: ACTRN12609000699268 Australian New Zealand Clinical Trials Registry. Date registered 13/Aug/2009 (prospective registration).
Assuntos
Anticoagulantes/administração & dosagem , Enoxaparina/administração & dosagem , Retardo do Crescimento Fetal/prevenção & controle , Pré-Eclâmpsia/prevenção & controle , Gravidez de Alto Risco/efeitos dos fármacos , Adulto , Protocolos Clínicos , Feminino , Idade Gestacional , Humanos , Recém-Nascido Pequeno para a Idade Gestacional , Gravidez , Resultado da Gravidez , Resultado do Tratamento , Adulto JovemRESUMO
Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialised topics. At the 2015 IFPA annual meeting there were 12 themed workshops, three of which are summarized in this report. These workshops related to various aspects of placental biology and collectively covered areas of obesity and the placenta, stem cells of the feto-maternal interface, and placental immunobiology and infection.
Assuntos
Obesidade/metabolismo , Doenças Placentárias/metabolismo , Placenta/metabolismo , Células-Tronco/metabolismo , Feminino , Humanos , GravidezRESUMO
The mechanisms by which the fetus induces maternal physiological adaptations to pregnancy are unclear. Cellular debris, shed from the placental syncytiotrophoblast into the maternal blood and phagocytosed by maternal endothelial and immune cells, may be one of these mechanisms. Here we show that trophoblastic debris from normal first trimester placentae induces changes in the transcriptome and proteome of endothelial cells in vitro, which might contribute to the adaptation of the maternal cardiovascular system to pregnancy. Trophoblastic debris also induced endothelial cells to transcribe placenta-specific genes, including the vasodilator hormone CSH1, thereby expanding the effective functional size of the placenta. Our data suggest that the deportation of trophoblastic debris is an important part of the complex network of feto-maternal communication.
Assuntos
Células Endoteliais/fisiologia , Perfilação da Expressão Gênica , Troca Materno-Fetal , Trofoblastos/fisiologia , Células Cultivadas , Feminino , Humanos , Gravidez , Proteoma/análiseRESUMO
BACKGROUND: Preeclampsia is a pregnancy-specific disorder characterised by an inappropriate maternal inflammatory response during pregnancy. High mobility group box 1 (HMGB1) was originally characterised as a nuclear protein but when released into the extracellular environment following necrotic cell death, it is proinflammatory. HMGB1 is expressed in the syncytiotrophoblast of human placenta. Higher levels of uric acid are reported in preeclampsia. The aim of this study was to investigate whether the expression of HMGB1differed between early onset and late onset preeclampsia or severe and mild preeclampsia and whether its expression correlated with the levels of uric acid. METHODS: 74 preeclamptic placentae and 110 normotensive placentae were included in this study. The levels of uric acid in women with preeclampsia were measured. The expression of HMGB1 in preeclamptic placentae or in first trimester and term placentae that had been treated with uric acid was measured. RESULTS: HMGB1 was expressed predominantly in the syncytiotrophoblast of the placenta and the expression of HMGB1 in the cytoplasm of the syncytiotrophoblast was significantly increased in both severe preeclampsia and early onset preeclampsia compared to normotensive pregnancies. The circulating levels of uric acid were significantly increased in preeclampsia and correlated with the expression of HMGB1. Increased levels of HMGB1 were significantly correlated with the severity and the time of onset of preeclampsia, but pathologic levels of uric acid did not increase the expression of HMGB1. CONCLUSION: Our data provides a better understanding of the function of HMGB1, a danger molecule in the pathogenesis of preeclampsia.
Assuntos
Citoplasma/metabolismo , Proteína HMGB1/metabolismo , Placenta/metabolismo , Trofoblastos/metabolismo , Adolescente , Adulto , Feminino , Humanos , Pré-Eclâmpsia/metabolismo , Gravidez , Ácido Úrico/metabolismo , Adulto JovemRESUMO
Early severe preeclampsia with changes consistent with the Hemolysis elevated liver enzymes low platelet count (HELLP) variant and severe fetal growth restriction rarely resolves prior to delivery. Established clinical disease is preceded by endothelial dysfunction and inflammation. Endothelial activation is reported in vitro to be raised in the presence of necrotic trophoblastic debris which is deported into the maternal circulation in preeclampsia. We report on an early severe preeclamptic patient admitted at 24 weeks gestation. Maternal serum was taken at day 2, 16, 30 of admission and 45 days postpartum. 20% maternal serum or trophoblastic debris from first trimester placental explants that had been cultured with 10% maternal serum was exposed to endothelial cells. Endothelial cell activation was quantified by the cell surface ICAM-1 expression and U937 monocyte adhesion assay. The clinical condition of this patient improved including the blood pressure, liver function, and platelet count by the 3rd day after antihypertensive treatment and remained normal until delivery at 37 weeks. ICAM-1 expression and U937 moncyte adhesion assay of endothelial cells was significantly increased following exposure of the endothelial cells to the maternal serum or trophoblastic debris from placentae treated with maternal serum drawn on day 2. However, ICAM-1 expression and the monocyte adhesion assay were significantly reduced following exposure of endothelial cells to maternal serum or trophoblastic debris from placenta treated with maternal serum drawn on day 16 or 30. Our data suggest unknown factor(s) in the maternal serum triggered endothelial cell activation when the clinical symptoms were present. The improvement in the clinical condition occurred along with the changes in endothelial cell activation.
Assuntos
Células Endoteliais/metabolismo , Placenta/metabolismo , Pré-Eclâmpsia/diagnóstico , Primeiro Trimestre da Gravidez/metabolismo , Adulto , Feminino , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Pré-Eclâmpsia/sangue , Pré-Eclâmpsia/metabolismo , Gravidez , Primeiro Trimestre da Gravidez/sangue , Índice de Gravidade de DoençaRESUMO
Nanoparticle tracking analysis (NTA) is commonly used to count and size nano-sized particles. A sample loading pump can be used to analyse a larger sample volume, but it is unclear whether accuracy is affected. Using a NanoSight NS300 with the manufacturer-supplied pump, we examined synthetic silica and latex microspheres, liposomes and placental extracellular vesicles at different flow speeds. Analysis at flow speeds of 20 or 50 significantly reduced the measured concentration and mean/modal size of particles, particularly for mono-dispersed samples. We identify sample flow speed as a crucial instrument setting which should be reported in all studies that use NTA.
Assuntos
Rastreamento de Células , Vesículas Extracelulares/fisiologia , Nanopartículas/análise , Placenta/ultraestrutura , Rastreamento de Células/métodos , Vesículas Extracelulares/química , Feminino , Citometria de Fluxo/métodos , Humanos , Lipossomos/análise , Lipossomos/química , Microesferas , Movimento , Tamanho da Partícula , Placenta/química , Placenta/citologia , Gravidez , Dióxido de Silício/químicaRESUMO
Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2015 there were twelve themed workshops, three of which are summarized in this report. These workshops covered areas of placental regulation and nutrient handling: 1) placental epigenetics; 2) placental mitochondrial function; 3) placental transport systems.
Assuntos
Epigênese Genética , Mitocôndrias/metabolismo , Placenta/metabolismo , Placentação/fisiologia , Animais , Transporte Biológico/fisiologia , Feminino , Humanos , GravidezRESUMO
Preeclampsia is a disorder of pregnancy characterized by endothelial activation. It is believed to be a response to a 'toxin(s)' from the placenta including trophoblastic debris and inflammatory cytokines. Calcium is known to reduce the risk of preeclampsia but the mechanism of its protective effect remains unknown. In this study, we investigated the potential mechanism(s) of calcium supplementation for preventing endothelial activation induced by trophoblastic debris. Trophoblastic debris was harvested from preeclamptic placentae and also from first-trimester placentae, which had been treated with preeclamptic sera. Endothelial cells were then cultured with trophoblastic debris in the presence of calcium. Endothelial activation was measured by quantifying endothelial cell-surface intercellular adhesion molecule-1 (ICAM-1) and by U937 monocyte adhesion to endothelial cells. The expression of ICAM-1 and U937 adhesion to endothelial cells were significantly reduced following exposure of endothelial cells to trophoblastic debris from preeclamptic placenta or from first-trimester placentae treated with preeclamptic sera in the presence of calcium compared with treatment without calcium. The expression of ICAM-1 was also significantly reduced following exposure of endothelial cells to trophoblastic debris with the nitric oxide donor or following treatment of endothelial cells with interleukin (IL)-1ß in the presence of calcium. Our study demonstrated that calcium supplementation prevented endothelial cell activation induced by trophoblastic debris from preeclamptic placentae. The nitric oxide synthase (NOS) pathway and anti-inflammatory effects are involved in the action of calcium on endothelial cell activation. These findings may suggest, at least in part, the protective mechanism of calcium supplementation on preeclampsia.
Assuntos
Cálcio/uso terapêutico , Meios de Cultivo Condicionados , Células Endoteliais/efeitos dos fármacos , Pré-Eclâmpsia/tratamento farmacológico , Adolescente , Adulto , Linhagem Celular , Suplementos Nutricionais , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Interleucina-1beta , Pessoa de Meia-Idade , Nitroprussiato , Gravidez , Adulto JovemRESUMO
INTRODUCTION: Necrotic but not apoptotic trophoblastic debris can induce endothelial cell activation but the mechanism by which endothelial cells distinguish apoptotic from necrotic debris is unclear. The NALP3 inflammasome is a pattern recognition receptor that macrophages employ to recognise "danger signals" in necrotic cell corpses. In this study, we hypothesized that endothelial cells can identify and respond to necrotic trophoblastic debris via the NALP3 inflammasome. METHODS: The effect of trophoblastic debris on endothelial expression of NALP3 inflammasome components was investigated using qRT-PCR, immunoassays and fluorescent caspase 1 activity assay. IL-1ß in was quantified by ELISA. Endothelial cell activation was measured by cell surface ICAM expression and monocytes adhesion assay. RESULTS: The NALP3 inflammasome was expressed in resting vascular endothelial cells and is involved in endothelial response to danger signals. However, exposure to necrotic trophoblastic debris did not significantly alter the expression of any of the three components of the NALP3 inflammasome at the mRNA level, nor was caspase-1 activation increased. Conditioned media from endothelial cells exposed to necrotic trophoblastic debris contained elevated levels of IL-1ß which was derived from the necrotic debris and which contributed to endothelial cell activation. DISCUSSION: Necrotic trophoblastic debris induced endothelial cell activation through the IL-1ß/IL-1R pathway. However, the NALP3 inflammasome in endothelial cells was not involved in this process.
Assuntos
Proteínas de Transporte/metabolismo , Células Endoteliais/metabolismo , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Placenta/metabolismo , Trofoblastos/metabolismo , Caspase 1/metabolismo , Linhagem Celular , Feminino , Humanos , Monócitos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Necrose/metabolismo , Gravidez , Transdução de Sinais/fisiologiaRESUMO
The placenta is responsible for all nutrient and gas exchange between mother and baby during pregnancy. The differentiation of specialised placental epithelial cells called trophoblasts is essential for placental function, but we understand little about how these populations arise. Mouse trophoblast stem cells have allowed us to understand many of the factors that regulate murine trophoblast lineage development, but the human placenta is anatomically very different from the mouse, and it is imperative to isolate a human trophoblast stem cell to understand human placental development. Here we have developed a novel methodology to isolate a Hoechst side-population of trophoblasts from early gestation placentae and compared their transcriptome to differentiated trophoblast populations (cytotrophoblasts and extravillous trophoblasts) using microarray technology. Side-population trophoblasts clustered as a transcriptomically distinct population but were more closely related to cytotrophoblasts than extravillous trophoblasts. Side-population trophoblasts up-regulated a number of genes characteristic of trophectoderm and murine trophoblast stem cells in comparison to cytotrophoblasts or extravillous trophoblasts and could be distinguished from both of these more mature populations by a unique set of 22 up-regulated genes, which were enriched for morphogenesis and organ development and the regulation of growth functions. Cells expressing two of these genes (LAMA2 and COL6A3) were distributed throughout the cytotrophoblast layer at the trophoblast/mesenchymal interface. Comparisons to previously published trophoblast progenitor populations suggest that the side-population trophoblasts isolated in this work are a novel human trophoblast population. Future work will determine whether these cells exhibit functional progenitor/stem cell attributes.
Assuntos
Diferenciação Celular , Separação Celular/métodos , Vilosidades Coriônicas/crescimento & desenvolvimento , Placenta/citologia , Placentação/fisiologia , Células-Tronco/citologia , Trofoblastos/citologia , Animais , Proliferação de Células , Células Cultivadas , Vilosidades Coriônicas/metabolismo , Feminino , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Técnicas Imunoenzimáticas , Camundongos , Placenta/metabolismo , Gravidez , Primeiro Trimestre da Gravidez , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/metabolismo , Trofoblastos/metabolismoRESUMO
INTRODUCTION: Women with preeclampsia have elevated levels of inflammatory cytokines including IL-6. IL-6, which is known to activate endothelial cells and induce the production of necrotic trophoblastic debris from the placenta, may be important in the pathogenesis of preeclampsia. MgSO4 is a major therapy for the prevention of seizures in preeclampsia but it has been suggested to also have anti-inflammatory and vasodilatory properties. METHODS: 22 pregnant women with preeclampsia and 68 normotensive controls were recruited and circulating IL-6 levels in these women were measured before MgSO4 and nifedipine treatment and after delivery. In addition, endothelial cells were treated with IL-6 or necrotic trophoblastic debris, generated from first trimester placental explants in the presence or absence of MgSO4in vitro, and cell-surface ICAM-1 was measured by ELISA. The levels of IL-6 in the culture medium were also measured. Furthermore nitric oxide synthetase activity in endothelial cells that had been treated with IL-6 was measured using l-NAME. RESULTS: Circulating levels of IL-6 in preeclampsia were reduced significantly following administration of MgSO4. In vitro, MgSO4 reversed the activation of endothelial cells induced by IL-6 but not by necrotic trophoblastic debris. The effect of MgSO4 in reversing the IL-6 induced activation of endothelial cells was not dependent upon nitric oxide synthetase. Treating placental explants with MgSO4 prevented the production of necrotic trophoblastic debris induced by IL-6. DISCUSSION: we demonstrated that IL-6 levels drop following treatment with MgSO4 and nifedipine in vivo, and have identified several mechanisms by which this positive effect on IL-6 may occur in vitro.
Assuntos
Interleucina-6/sangue , Sulfato de Magnésio/uso terapêutico , Nifedipino/uso terapêutico , Pré-Eclâmpsia/sangue , Pré-Eclâmpsia/tratamento farmacológico , Adolescente , Adulto , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Feminino , Humanos , Interleucina-6/farmacologia , Óxido Nítrico Sintase/metabolismo , Gravidez , Resultado do Tratamento , Adulto JovemRESUMO
The human placenta is an anatomically unique structure that extrudes a variety of extracellular vesicles into the maternal blood (including syncytial nuclear aggregates, microvesicles, and nanovesicles). Large quantities of extracellular vesicles are produced by the placenta in both healthy and diseased pregnancies. Since their first description more than 120 years ago, placental extracellular vesicles are only now being recognized as important carriers for proteins, lipids, and nucleic acids, which may play a crucial role in feto-maternal communication. Here, we summarize the current literature on the cargos of placental extracellular vesicles and the known effects of such vesicles on maternal cells/systems, especially those of the maternal immune and vascular systems.
Assuntos
Vesículas Extracelulares/fisiologia , Placenta/fisiologia , Feminino , Humanos , Lipídeos , Troca Materno-Fetal/imunologia , Ácidos Nucleicos , Pré-Eclâmpsia/fisiopatologia , GravidezRESUMO
BACKGROUND: Antiphospholipid antibodies (aPL) are a family of auto-antibodies that are associated with an increased risk of recurrent miscarriage, intrauterine growth restriction and preterm birth. The placenta is a major target of aPL and it is likely that these antibodies promote pregnancy morbidity by affecting trophoblast function. Numerous studies have investigated the effect of aPL on trophoblast function in vitro. However, different trophoblast models and a variety of culture conditions have been employed, resulting in a myriad of different reported findings. This review systematically summarized those published studies that have investigated the effect of aPL on trophoblast function in vitro. In addition, the reported effects of pharmacological treatment on trophoblast function in the presence of aPL were also systematically reviewed. METHODS: PubMed, Scopus, Embase and Web of Science databases were searched using the keywords 'placenta OR trophoblast' AND 'antiphospholipid antibody OR antiphospholipid syndrome' up to 25 April 2014. Studies were excluded based on the absence of appropriate controls. The effects of aPL on trophoblast proliferation, death, syncytialization, invasion, hormone production, cytokine production, coagulation and complement activation were recorded. The effects of different treatments on the function of trophoblasts in the presence of aPL were also recorded. RESULTS: A total of 1071 records were retrieved from the four databases. After removing duplicates, the titles and abstracts of 529 articles were reviewed. Of those, 48 articles were read and relevant experimental results were extracted from 47 articles. CONCLUSIONS: This systematic review provides an overview of all the studies performed to date on the effects of aPL on trophoblast function in vitro. There is considerable support for aPL decreasing trophoblast viability, syncytialization and invasion in vitro. Some work has also suggested that aPL may affect the production of hormones and signalling molecules by trophoblasts, and may stimulate coagulation and complement activation in vitro. Current reports of the in vitro effects of therapeutic treatments on trophoblast function in the presence of aPL are inconclusive. This systematic review has highlighted many gaps in our knowledge of how aPL work and may direct future research in this area.
Assuntos
Anticorpos Antifosfolipídeos/fisiologia , Placenta/citologia , Placenta/imunologia , Trofoblastos/fisiologia , Síndrome Antifosfolipídica/tratamento farmacológico , Síndrome Antifosfolipídica/imunologia , Coagulação Sanguínea/fisiologia , Células Cultivadas , Ativação do Complemento/fisiologia , Feminino , Humanos , Técnicas In Vitro , Gravidez , Trofoblastos/citologiaRESUMO
INTRODUCTION: Preeclampsia is characterized by maternal endothelial dysfunction. While the mechanisms leading to preeclampsia are unclear, a factor(s) from the placenta is responsible for triggering the disease. One placental factor implicated in triggering preeclampsia is trophoblast debris which may transmit pathogenic signals from the placenta to endothelial cells. In this study, we investigated whether trophoblast debris from preeclamptic placentae triggered endothelial cell activation. METHODS: Trophoblast debris from preeclamptic or normotensive placentae, or trophoblast debris from normal placental explants that had been cultured with preeclamptic (n = 14) or normotensive sera (n = 14) was exposed to endothelial cells. Activation of the endothelial cells was quantified by cell surface ICAM-1 and U937 adhesion to endothelial cells. The levels of IL-1ß, pro-caspase-1 and active caspase-1 in the trophoblast debris were measured. RESULTS: Compared to controls, the levels of ICAM-1 and U937 adhesion to endothelial cells were significantly increased following exposure of the endothelial cells to trophoblast debris from preeclamptic placentae or placentae treated with preeclamptic sera. The levels IL-1ß, pro-caspase-1 and active caspase-1 were significantly increased in both trophoblast debris from preeclamptic placentae and placentae treated with preeclamptic sera. DISCUSSION: These results provide the first direct evidence that trophoblast debris produced from preeclamptic placentae or placentae treated with preeclamptic sera can activate the endothelium. CONCLUSIONS: Trophoblast debris from preeclamptic but not normotensive placentae can induce endothelial cell activation. This may be one mechanism by which the preeclamptic placenta communicates with the maternal endothelium to induce activation of the endothelium.
Assuntos
Comunicação Celular/fisiologia , Células Endoteliais/metabolismo , Placenta/metabolismo , Pré-Eclâmpsia/metabolismo , Trofoblastos/metabolismo , Caspase 1/metabolismo , Células Cultivadas , Endotélio/metabolismo , Feminino , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , GravidezRESUMO
BACKGROUND: During the first trimester of human pregnancy, specialised placental cells called extravillous trophoblasts (EVTs) grow out from anchoring villi, invade the maternal decidua and remodel the uterine spiral arteries. Inadequate EVT invasion is associated with pregnancy complications including intrauterine growth restriction (IUGR) and pre-eclampsia. During early pregnancy, the placenta exists in a physiologically normal low oxygen environment, which may regulate EVT outgrowth. One potential oxygen responsive regulator of EVTs is the transforming growth factor-beta (TGFß) family of cytokines. This work aimed to determine the role of TGFß1, ß2 and ß3 in regulating EVT outgrowth in the low oxygen environment of early pregnancy. RESULTS: Using a quantitative high-throughput first trimester villous explant model of EVT outgrowth we demonstrated no significant difference in the frequency of EVT outgrowth between explants treated with TGFß1, ß2 or ß3. However, explants treated with TGFß2, but not ß1 or ß3, produced EVT outgrowths with a significantly smaller area in comparison to untreated controls (p=0.03). When explants were cultured in 1.5% oxygen, TGFß2, but not ß1 or ß3, in the conditioned medium of explants that produced EVT outgrowth was significantly reduced in comparison to 8% oxygen (p<0.05). There was no significant difference in the concentration of TGFß2 or TGFß3 from isolated primary EVTs cultured in 1.5% or 8% oxygen. CONCLUSIONS: TGFß2 inhibits EVT outgrowth expansion from first trimester anchoring villi. As TGFß2 secretion from anchoring villi is down-regulated in low oxygen, these findings suggest that the low oxygen environment in early pregnancy may be important to allow EVT outgrowth expansion and promote adequate placentation.
Assuntos
Vilosidades Coriônicas/metabolismo , Oxigênio/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Trofoblastos/citologia , Meios de Cultivo Condicionados , Feminino , Retardo do Crescimento Fetal , Humanos , Técnicas In Vitro , Gravidez , Primeiro Trimestre da Gravidez , Trofoblastos/metabolismoRESUMO
Immunoelectron microscopy is wrought with technical limitations that complicate its use. However, advances in correlative light and electron microscopy have recently lead to improvements in this field. We report the development of a semi-correlative approach to investigate the ultrastructural location of an antiphospholipid antibody within the syncytiotrophoblast. This method offers several advantages over existing methodologies, since it preserves antigenicity, shows good immunolabel penetrability and does not require specialized equipment. The use of a pre-embedding screen has also allowed us to target individual placental villi and overcome sampling limitations of the electron microscope. This simple, cost-effective method is likely to find widespread application in placental research.
Assuntos
Microscopia Eletrônica/métodos , Microscopia Imunoeletrônica/métodos , Proteínas da Gravidez/metabolismo , Trofoblastos/química , Animais , Anticorpos Antifosfolipídeos/análise , Feminino , Humanos , GravidezRESUMO
In pregnancy disorders such as pre-eclampsia, intrauterine growth restriction (IUGR) and recurrent miscarriage a poorly functioning placenta is thought to be a major component of the disease process. However, despite their prevalence, we currently have no way to fix dysfunctional placentae or directly treat these disorders. Over the past two decades our understanding of the role that stem cells play in organ development and regeneration has expanded rapidly, and over the past 5 years the therapeutic use of stem cells to both regenerate damaged tissues, and act as potent modulators of diseased microenvironments, has become a reality in many organs including the heart, kidney, liver, skin and eye. Over its short lifespan the placenta undergoes rapid and continuous growth and differentiation, meaning that placental 'organogenesis' only truly ends at delivery, and thus stem cells are likely to play important roles in placental function for the duration of pregnancy. Two populations of stem cells exist in the placenta that contribute to this on-going growth and differentiation: trophoblast stem cells and mesenchymal stem cells. This review will address our current understanding of how each of these stem cell populations contributes to successful placental function, how epithelial and mesenchymal stem cell populations are being translated to the clinic in other fields, and whether these advances can teach us anything about how placental stem cells could be used to fix faulty placentae in the future.