RESUMEN
During pregnancy, apoptosis is a physiological event critical in the remodeling and aging of the placenta. Increasing evidence has pointed toward the relevance of hypoxia as modulator of trophoblast cell death. Previous reports have shown that leptin, a placental cytokine, promotes cell survival in both cell culture and placental explant models. The aim of this work is to establish the role of leptin in apoptosis under hypoxic condition in trophoblast cells. In this study, we evaluated the effect of cobalt chloride, a hypoxia mimicking agent that stabilizes the expression of hypoxia-inducible factor-1 alpha, on Swan-71 and human placental explants. Hypoxia chamber was also used to generate 2% oxygen. Apoptosis was determined by the presence of apoptotic nucleus, fragmentation of DNA and Caspase-3 and PARP-1 cleavage. The pro-apoptotic proteins BAX, BID, BAD, and BAK and the anti-apoptotic effectors BCL-2, B-cell lymphoma-extra-large, and myeloid cell leukemia-1 were also analyzed. We found that hypoxia-inducible factor-1 alpha stabilization increased the appearance of apoptotic nucleus, fragmentation of DNA, and Caspase-3 and PARP-1 cleavage. Hypoxia mimicking conditions enhanced the expression of pro-apoptotic effectors BAX, BID, BAD, and BAK. Hypoxia-inducible factor-1 alpha stabilization also downregulated the level of BCL-2, B-cell lymphoma-extra-large, and myeloid cell leukemia-1. All these apoptotic parameters changes were reversed with leptin treatment. Moreover, we showed that leptin action on apoptosis modulation involves PI3K and MAPK signaling pathways. Obtained data demonstrate that hypoxia-inducible factor-1 alpha stabilization induces apoptosis in human placenta and leptin counteracts this effect, reinforcing its role as a survival cytokine.
Asunto(s)
Apoptosis , Leptina , Placenta , Humanos , Femenino , Placenta/metabolismo , Placenta/efectos de los fármacos , Embarazo , Leptina/metabolismo , Leptina/farmacología , Apoptosis/efectos de los fármacos , Trofoblastos/metabolismo , Trofoblastos/efectos de los fármacos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Cobalto/farmacología , Hipoxia de la Célula/fisiologíaRESUMEN
Adverse pregnancy outcomes have been associated with the presence of glyphosate (G) in umbilical cord, serum, and urine samples from pregnant women. Our aim was to study the effect of G on blastocyst implantation using an in vitro mouse model, and the migration and acquisition of endothelial phenotype of the human trophoblastic HTR8/SVneo (H8) cells. In mouse blastocysts, no differences in attachment time and implantation outgrowth area were observed after G exposure. H8 cell migration was stimulated by 0.625 µM G without cytotoxicity. After 6 h, the mRNA expression of vascular endothelial growth factor (VEGF) and C-C motif chemokine ligand 2 (CCL2) was upregulated in H8 cells exposed to 1.25 µM G when compared vehicle-treated cells (p ≤ 0.05). No differences were observed in interleukin 11, VEGF receptor 1, and coagulation factor II thrombin receptor in H8 cells exposed to different concentrations of G for 6 h compared to the vehicle. Interestingly, exposure to G did not alter angiogenesis as measured by a tube formation assay. Taken all together, these results suggest that G exposure may contribute as a risk factor during pregnancy, due to its ability to alter trophoblast migration and gene expression.
Asunto(s)
Blastocisto , Movimiento Celular , Implantación del Embrión , Glicina , Glifosato , Trofoblastos , Trofoblastos/efectos de los fármacos , Trofoblastos/metabolismo , Movimiento Celular/efectos de los fármacos , Humanos , Animales , Femenino , Ratones , Glicina/análogos & derivados , Glicina/toxicidad , Glicina/farmacología , Blastocisto/efectos de los fármacos , Blastocisto/metabolismo , Implantación del Embrión/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Línea Celular , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Embarazo , Herbicidas/toxicidad , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , AngiogénesisRESUMEN
Benzophenone-3 (BP3) is a common ingredient in personal care products (PCPs) due to its well-established effectiveness in absorbing UV radiation. Sunscreen products are among the most widely used PCPs-containing BP3 applied to the skin, resulting in significant human exposure to BP3 primarily through a dermal application. In the present work, we have tested the action of three environmentally relevant concentrations of BP3 (2, 20 and 200 µg/L) on an in vitro model of implantation of murine blastocysts and on migration ability of the human trophoblast cell line Swan 71. We showed that BP3 caused a significant reduction of blastocyst expansion and a delayed hatching in a non-monotonic way. Besides, embryos displayed a delayed attachment in the three BP3 groups, resulting in a smaller implantation area on the 6th day of culture: BP3(2) (0.32 ± 0.07 mm2); BP3(20) (0.30 ± 0.08 mm2) and BP3(200) (0.25 ± 0.06 mm2) in comparison to the control (0.42 ± 0.07 mm2). We also found a reduced migration capacity of the human first-trimester trophoblast cell line Swan 71 in a scratch assay when exposed to BP3: the lowest dose displayed a higher uncovered area (UA) at 6h when compared to the control, whereas a higher UA of the wound was observed for the three BP3 concentrations at 18 and 24 h of exposure. The changes in UA provoked by BP3 restored to normal values in the presence of flutamide, an androgen receptor (AR) inhibitor. These results indicate that a direct impairment on early embryo implantation and a defective migration of extravillous trophoblast cells through the androgen receptor pathway can be postulated as mechanisms of BP3-action on early gestation with potential impact on fetal growth.
Asunto(s)
Benzofenonas , Movimiento Celular , Implantación del Embrión , Protectores Solares , Trofoblastos , Rayos Ultravioleta , Benzofenonas/toxicidad , Protectores Solares/toxicidad , Protectores Solares/farmacología , Trofoblastos/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Ratones , Animales , Humanos , Implantación del Embrión/efectos de los fármacos , Blastocisto/efectos de los fármacos , Femenino , Línea CelularRESUMEN
Crosstalk between trophoblast and monocytes is essential for gestational success, and it can be compromised in congenital toxoplasmosis. Cell death is one of the mechanisms involved in the maintenance of pregnancy, and this study aimed to evaluate the role of trophoblast in the modulation of monocyte cell death in the presence or absence of Toxoplasma gondii infection. THP-1 cells were stimulated with supernatants of BeWo cells and then infected or not with T. gondii. The supernatants were collected and analyzed for the secretion of human Fas ligand, and cells were used to determine cell death and apoptosis, cell death receptor, and intracellular proteins expression. Cell death and apoptosis index were higher in uninfected THP-1 cells stimulated with supernatants of BeWo cells; however, apoptosis index was reduced by T. gondii infection. Macrophage migration inhibitory factor (MIF) and transforming growth factor (TGF)-ß1, secreted by BeWo cells, altered the cell death and apoptosis rates in THP-1 cells. In infected THP-1 cells, the expression of Fas/CD95 and secretion of FasL was significantly higher; however, caspase 3 and phosphorylated extracellular-signal-regulated kinase (ERK1/2) were downregulated. Results suggest that soluble factors secreted by BeWo cells induce cell death and apoptosis in THP-1 cells, and Fas/CD95 can be involved in this process. On the other hand, T. gondii interferes in the mechanism of cell death and inhibits THP-1 cell apoptosis, which can be associated with active caspase 3 and phosphorylated ERK1/2. In conclusion, our results showed that human BeWo trophoblast cells and T. gondii infection modulate cell death in human THP-1 monocyte cells.
Asunto(s)
Espacio Intracelular/metabolismo , Monocitos/patología , Monocitos/parasitología , Proteínas/metabolismo , Receptores de Muerte Celular/metabolismo , Toxoplasmosis/patología , Trofoblastos/parasitología , Caspasa 3/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular , Medios de Cultivo Condicionados/farmacología , Regulación hacia Abajo/efectos de los fármacos , Proteína Ligando Fas/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Factores Inhibidores de la Migración de Macrófagos/farmacología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Fosforilación/efectos de los fármacos , Células THP-1 , Trofoblastos/efectos de los fármacos , Trofoblastos/metabolismo , Receptor fas/metabolismoRESUMEN
Preeclampsia and intrauterine growth restriction, multisystemic disorders characterized by a shallow trophoblast invasion, have been associated with maternal cadmium (Cd) exposure. The molecular mechanisms of this association remain unknown. Cell adhesion and matrix metalloproteinase production are essential for an adequate trophoblast invasion. Thus, the aim of this study was to determine the effect of Cd exposure on invasion, adhesion, and matrix metalloproteinase-9 (MMP-9) production in the trophoblast-derived HTR-8/SVneo cell line. Cultured HTR-8/SVneo trophoblast cells were incubated with different concentrations of CdCl2 for 6 h. Cell invasion was determined by the transwell assay, while cell adhesion was examined on collagen type I. MMP-9 release and activity were measured by ELISA and zymography, respectively. MMP-9 mRNA expression was detected by reverse-transcription polymerase chain reaction, while intracellular MMP-9 protein was assessed by Western blotting. Cd exposure significantly decreased the invasion and adhesion of HTR-8/SVneo cells. Also, MMP-9 levels and activity in the culture medium were significantly reduced after Cd incubation. In contrast, MMP-9 mRNA expression and intracellular protein levels were significantly increased. These data indicate that Cd reduces trophoblast cells invasiveness by inhibiting cell adhesion and MMP-9 secretion.
Asunto(s)
Cadmio/farmacología , Adhesión Celular/efectos de los fármacos , Metaloproteinasa 2 de la Matriz/metabolismo , Trofoblastos/fisiología , Cloruro de Cadmio/administración & dosificación , Línea Celular , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Exposición Materna , Metaloproteinasa 2 de la Matriz/análisis , Metaloproteinasa 2 de la Matriz/genética , Embarazo , ARN Mensajero/análisis , Trofoblastos/efectos de los fármacosRESUMEN
BACKGROUND AND AIM: The endoplasmic reticulum (ER) stress response and the unfolded protein response (UPR) are essential cellular mechanisms to ensure the proper functioning of ER in adverse conditions. However, activation of these pathways has also been associated with insulin resistance and cell death in pathological conditions such as diabetes mellitus. In the present study, we investigated whether stromal cell-derived factor 2 (SDF2)-an ER stress-responsive factor-is related to ER response in placental cells exposed to maternal gestational diabetes mellitus (GDM) or to a hyperglycaemic in vitro condition. OBJECTIVE: The study aimed to investigate the role of SDF2 in BeWo cells , a trophoblast cell line originating from choriocarcinoma , and in placental tissue under hyperglycaemic conditions. METHODS: Protein levels of SDF2 and UPR factors, glucose-related protein 78 (GRP78) and eukaryotic initiation factor 2 alpha (elF2 alpha) were evaluated in the placentae of pregnant women diagnosed with GDM and treated by diet-control (insulin was added when necessary). The mRNA expression of SDF2 and UPR factors CHOP and sXBP1 were assessed in cultured BeWo cells challenged with glucose and treated with or without insulin. RESULTS: SDF2 expression was increased in the placentae of GDM women treated with diet. However, its values were similar to those of normoglycemic controls when the GDM women were treated with insulin and diet. BeWo cells cultured with high glucose and insulin showed decreased SDF2 expression, while high glucose increased CHOP and sXBP1 expression, which was then significantly reverted with insulin treatment. CONCLUSION: Our findings extend the understanding of ER stress and SDF2 expression in placentae exposed to hyperglycaemia, highlighting the relevance of insulin in reducing the levels of ER stress factors in placental cells. Understanding the effect of ER stress partners such as SDF2 on signalling pathways involved in gestation, complicated by hyperglycaemia, is pivotal for basic biomedical research and may lead to new therapeutic possibilities.
Asunto(s)
Glucemia/metabolismo , Diabetes Gestacional/metabolismo , Estrés del Retículo Endoplásmico , Proteínas/metabolismo , Trofoblastos/metabolismo , Estudios de Casos y Controles , Línea Celular Tumoral , Estudios Transversales , Diabetes Gestacional/sangre , Diabetes Gestacional/patología , Diabetes Gestacional/terapia , Dieta Saludable , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Factor 2 Eucariótico de Iniciación/metabolismo , Femenino , Proteínas de Choque Térmico/metabolismo , Humanos , Hipoglucemiantes/uso terapéutico , Insulina/uso terapéutico , Embarazo , Proteínas/genética , Transducción de Señal , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismo , Trofoblastos/efectos de los fármacos , Trofoblastos/patología , Proteína 1 de Unión a la X-Box/genética , Proteína 1 de Unión a la X-Box/metabolismoRESUMEN
The combination of pyrimethamine and sulfadiazine is the standard care in cases of congenital toxoplasmosis. However, therapy with these drugs is associated with severe and sometimes life-threatening side effects. The investigation of phytotherapeutic alternatives to treat parasitic diseases without acute toxicity is essential for the advancement of current therapeutic practices. The present study investigates the antiparasitic effects of oleoresins from different species of Copaifera genus against T. gondii. Oleoresins from C. reticulata, C. duckei, C. paupera, and C. pubiflora were used to treat human trophoblastic cells (BeWo cells) and human villous explants infected with T. gondii. Our results demonstrated that oleoresins were able to reduce T. gondii intracellular proliferation, adhesion, and invasion. We observed an irreversible concentration-dependent antiparasitic action in infected BeWo cells, as well as parasite cell cycle arrest in the S/M phase. The oleoresins altered the host cell environment by modulation of ROS, IL-6, and MIF production in BeWo cells. Also, Copaifera oleoresins reduced parasite replication and TNF-α release in villous explants. Anti-T. gondii effects triggered by the oleoresins are associated with immunomodulation of the host cells, as well as, direct action on parasites.
Asunto(s)
Antiprotozoarios/farmacología , Fabaceae/química , Extractos Vegetales/farmacología , Complicaciones Parasitarias del Embarazo/tratamiento farmacológico , Toxoplasmosis/complicaciones , Toxoplasmosis/tratamiento farmacológico , Animales , Antiprotozoarios/administración & dosificación , Antiprotozoarios/aislamiento & purificación , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Fabaceae/clasificación , Femenino , Interacciones Huésped-Parásitos/efectos de los fármacos , Humanos , Microscopía Electrónica de Transmisión , Fitoterapia , Placenta/efectos de los fármacos , Placenta/parasitología , Extractos Vegetales/administración & dosificación , Extractos Vegetales/aislamiento & purificación , Embarazo , Complicaciones Parasitarias del Embarazo/parasitología , Especies Reactivas de Oxígeno/metabolismo , Toxoplasma/citología , Toxoplasma/efectos de los fármacos , Toxoplasma/patogenicidad , Toxoplasmosis/parasitología , Trofoblastos/efectos de los fármacos , Trofoblastos/parasitologíaRESUMEN
BACKGROUND: More than 3 years since the last Zika virus (ZIKV) outbreak in Brazil, researchers are still deciphering the molecular mechanisms of neurovirulence and vertical transmission, as well as the best way to control spread of ZIKV, a flavivirus. The use of pesticides was the main strategy of mosquito control during the last ZIKV outbreak. METHODS: We used vesicular stomatitis virus (VSV) tagged with green fluorescent protein (GFP) as our prototypical virus to study the impact of insecticide pyriproxyfen (PPF). VZV-GFP infected and uninfected Jurkat, HeLa and trophoblast cells were treated with PPF and compared to untreated cells (control). Cell viability was determined by the MTT assay. Cell morphology, presence of extracellular vesicles (EVs), virus infection/GFP expression as well as active mitochondrial levels/localization were examined by confocal microscopy. RESULTS: PPF, which was used to control mosquito populations in Brazil prior to the ZIKV outbreak, enhances VSV replication and has cell membrane-altering properties in the presence of virus. PPF causes enhanced viral replication and formation of large EVs, loaded with virus as well as mitochondria. Treatment of trophoblasts or HeLa cells with increasing concentrations of PPF does not alter cell viability, however, it proportionately increases Jurkat cell viability. Increasing concentrations of PPF followed by VSV infection does not interfere with HeLa cell viability. Both Jurkats and trophoblasts show proportionately increased cell death with increased concentrations of PPF in the presence of virus. CONCLUSIONS: We hypothesize that PPF disrupts the lipid microenvironment of mammalian cells, thereby interfering with pathways of viral replication. PPF lowers viability of trophoblasts and Jurkats in the presence of VSV, implying that the combination renders immune system impairment in infected individuals as well as enhanced vulnerability of fetuses towards viral vertical transmission. We hypothesize that similar viruses such as ZIKV may be vertically transmitted via EV-to-cell contact when exposed to PPF, thereby bypassing immune detection. The impact of pesticides on viral replication must be fully investigated before large scale use in future outbreaks of mosquito borne viruses.
Asunto(s)
Infecciones por Flavivirus/transmisión , Insecticidas/farmacología , Piridinas/farmacología , Vesiculovirus/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Aedes/virología , Animales , Brasil , Supervivencia Celular/efectos de los fármacos , Virus del Dengue/efectos de los fármacos , Vesículas Extracelulares/efectos de los fármacos , Vesículas Extracelulares/virología , Proteínas Fluorescentes Verdes/genética , Células HeLa , Humanos , Células Jurkat , Trofoblastos/efectos de los fármacos , Trofoblastos/virología , Virulencia , Virus Zika/efectos de los fármacosRESUMEN
INTRODUCTION: The pleiotropic kininogen-kallikrein-kinin system is upregulated in pregnancy and localizes in the uteroplacental unit. To identify the systemic and local participation of the bradykinin type 2 receptor (B2R), this was antagonized by Bradyzide (BDZ) during 2 periods: from days 20 to 34 and from days 20 to 60 in pregnant guinea pigs. METHODS: Pregnant guinea pigs received subcutaneous infusions of saline or BDZ from gestational day 20 until sacrifice on day 34 (Short B2R Antagonism [SH-B2RA]) or on day 60 (Prolonged B2R Antagonism [PR-B2RA]). In SH-BDZA, systolic blood pressure was determined on day 34, while in PR-BDZA it was measured preconceptionally, at days 40 and 60. On gestational day 60, plasma creatinine, uricemia, proteinuria, fetal, placental and maternal kidney weight, and the extent of trophoblast invasion were evaluated. RESULTS: The SH-B2RA increased systolic blood pressure on day 34 and reduced trophoblast myometrial invasion, spiral artery remodeling, and placental sufficiency. The PR-B2RA suppressed the normal blood pressure fall observed on days 40 and 60; vascular transformation, placental efficiency, urinary protein, serum creatinine, and uric acid did not differ between the groups. The proportion of all studied mothers with lost fetuses was greater under BDZ infusion than in controls. CONCLUSION: The increased systolic blood pressure and transient reduction in trophoblast invasion and fetal/placental weight in the SH-B2R blockade and the isolated impact on blood pressure in the PR-B2R blockade indicate that bradykinin independently modulates systemic hemodynamics and the uteroplacental unit through cognate vascular and local B2R receptors.
Asunto(s)
Presión Sanguínea/fisiología , Antagonistas de los Receptores de Bradiquinina/farmacología , Bradiquinina/metabolismo , Receptor de Bradiquinina B2/metabolismo , Trofoblastos/metabolismo , Animales , Presión Sanguínea/efectos de los fármacos , Bradiquinina/antagonistas & inhibidores , Femenino , Cobayas , Placenta/efectos de los fármacos , Placenta/metabolismo , Embarazo , Pirrolidinas/farmacología , Tiosemicarbazonas/farmacología , Trofoblastos/efectos de los fármacosRESUMEN
Placental malaria (PM) is associated with severe inflammation leading to abortion, preterm delivery, and intrauterine growth restriction. Innate immunity responses play critical roles, but the mechanisms underlying placental immunopathology are still unclear. Here, we investigated the role of inflammasome activation in PM by scrutinizing human placenta samples from an endemic area and ablating inflammasome components in a PM mouse model. The reduction in birth weight in babies from infected mothers is paralleled by increased placental expression of AIM2 and NLRP3 inflammasomes. Using genetic dissection, we reveal that inflammasome activation pathways are involved in the production and detrimental action of interleukin-1ß (IL-1ß) in the infected placenta. The IL-1R pharmacological antagonist Anakinra improved pregnancy outcomes by restoring fetal growth and reducing resorption in an experimental model. These findings unveil that IL-1ß-mediated signaling is a determinant of PM pathogenesis, suggesting that IL-1R antagonists can improve clinical outcomes of malaria infection in pregnancy.
Asunto(s)
Inflamasomas/efectos de los fármacos , Interleucina-1beta/inmunología , Malaria Falciparum/inmunología , Malaria/inmunología , Plasmodium falciparum/patogenicidad , Complicaciones Parasitarias del Embarazo/inmunología , Transducción de Señal/efectos de los fármacos , Animales , Caspasa 1/genética , Caspasa 1/inmunología , Línea Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Femenino , Regulación de la Expresión Génica , Humanos , Inmunidad Innata , Factores Inmunológicos/farmacología , Inflamasomas/genética , Inflamasomas/inmunología , Interferón gamma/genética , Interferón gamma/inmunología , Proteína Antagonista del Receptor de Interleucina 1/farmacología , Interleucina-1beta/antagonistas & inhibidores , Interleucina-1beta/genética , Malaria/tratamiento farmacológico , Malaria/genética , Malaria/parasitología , Malaria Falciparum/genética , Malaria Falciparum/parasitología , Malaria Falciparum/patología , Ratones , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Plasmodium berghei/inmunología , Plasmodium berghei/patogenicidad , Plasmodium falciparum/inmunología , Embarazo , Complicaciones Parasitarias del Embarazo/genética , Complicaciones Parasitarias del Embarazo/parasitología , Complicaciones Parasitarias del Embarazo/prevención & control , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/inmunología , Transducción de Señal/inmunología , Células THP-1 , Trofoblastos/efectos de los fármacos , Trofoblastos/inmunología , Trofoblastos/parasitología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
BACKGROUND: Group B streptococcus (GBS) is the main bacteria that infects pregnant women and can cause abortion and chorioamnionitis. The impact of GBS effects on human trophoblast cells remains largely elusive, and actions toward anti-inflammatory strategies in pregnancy are needed. A potent anti-inflammatory molecule, uvaol is a triterpene from olive oil and its functions in trophoblasts are unknown. We aimed to analyze biomechanical and functional effects of inactivated GBS in trophoblast cells, with the addition of uvaol to test potential benefits. METHODS: HTR-8/SVneo cells were treated with uvaol and incubated with inactivated GBS. Cell viability and death were analyzed. Cellular elasticity and topography were accessed by atomic force microscopy. Nitrite production was evaluated by Griess reaction. Nuclear translocation of NFkB p65 was detected by immunofluorescence and Th1/Th2 cytokines by bead-based multiplex assay. RESULTS: GBS at 108â¯CFU increased cell death, which was partially prevented by uvaol. Cell stiffness, cytoskeleton organization and morphology were changed by GBS, and uvaol partially restored these alterations. Nuclear translocation of NFkB p65 began 15â¯min after GBS incubation and uvaol inhibited this process. GBS decreased IL-4 secretion and increased IL-1ß, IFN-γ and IL-2, whereas uvaol reverted this. CONCLUSIONS: The increased inflammation and cell death caused by GBS correlated with biomechanical and cytoskeleton changes found in trophoblast cells, while uvaol was effective its protective role. GENERAL SIGNIFICANCE: Uvaol is a natural anti-inflammatory product efficient against GBS-induced inflammation and it has potential to be acquired through diet in order to prevent GBS deleterious effects in pregnancy.
Asunto(s)
Streptococcus agalactiae/patogenicidad , Triterpenos/farmacología , Trofoblastos/efectos de los fármacos , Trofoblastos/microbiología , Animales , Transporte Biológico , Fenómenos Biomecánicos , Muerte Celular , Línea Celular , Núcleo Celular/metabolismo , Supervivencia Celular , Chlorocebus aethiops , Citocinas/metabolismo , Femenino , Humanos , FN-kappa B/metabolismo , Nitritos/metabolismo , Embarazo , Células TH1/metabolismo , Trofoblastos/metabolismo , Células VeroRESUMEN
Shiga toxin (Stx2) producing Escherichia coli infections during early gestation may impair placentation through a Stx2 damage of extravillous trophoblast (EVT) cells. We have previously demonstrated that Stx2 injected in rats in the early stage of pregnancy causes spontaneous abortion by a direct cytotoxic effect in the highly perfused feto-uteroplacental unit. The main aim was to evaluate the effects of Stx2 on EVT in order to understand the possible adverse effects that the toxin may have on trophoblast cells during early pregnancy. Swan 71 and HTR-8 cell lines were used as human EVT models. The presence of Stx2 receptor, globotriaosylceramide (Gb3), on Swan 71 and HTR-8 cells was evaluated by thin layer chromatography. The effects of Stx2 on cell viability were evaluated by neutral red uptake, migration by wound-healing assay and invasion was determined by the 'transwell chamber' assay. Metalloproteinase activity (MMP-2) was evaluated by zymography and tubulogenesis was analyzed by counting the total tube length and the number of branch formation. We have demonstrated that Swan 71 expresses high levels of Gb3 compared to HTR-8 cells. Stx2 decreased significantly Swan 71 viability in a dose-dependent manner after 72 h of toxin exposure. Furthermore, Stx2 impaired migration, invasion and tube-like formation of Swan 71 cells and decreased the MMP-2 activity. These cytotoxic effects were partially prevented by aminoguanidine, an inducible nitric oxide synthase inhibitor. These studies demonstrate that the function and viability of EVT cells may be altered by Stx2 and suggest that NO overexpression may be involved in the detrimental effects.
Asunto(s)
Movimiento Celular , Supervivencia Celular , Toxina Shiga II/efectos adversos , Trofoblastos/patología , Células Cultivadas , Femenino , Humanos , Embarazo , Trihexosilceramidas/metabolismo , Trofoblastos/efectos de los fármacos , Trofoblastos/metabolismoRESUMEN
Spiral artery remodeling at the maternal-fetal interface is crucial for successful pregnancy and requires the interaction between the first trimester trophoblast and the endothelial cells of the maternal vessels. However, the precise mechanism of this dialog has yet to be determined. The current study investigated whether lysophosphatidic acid (LPA) modulates trophoblast-endothelial crosstalk in vitro. HTR-8/SVneo trophoblast cell line (H8) was seeded on top of Geltrex, incubated with LPA or LPA + NS-398 (selective cyclooxygenase-2 inhibitor), LPA + 1400W (selective inducible nitric oxide synthase inhibitor) or LPA + IL-6 neutralizing antibody and assayed for tube formation to model the acquisition of trophoblast endovascular phenotype. The supernatants were collected and used as conditioned media (CM). To test trophoblast-endothelial crosstalk, the endothelial cell line EA.hy926 was incubated with trophoblast CM. The CM from LPA-induced tubulogenesis stimulated endothelial cells migration and did not modify the apoptosis. Soluble factors derived from cyclooxygenase-2 and IL-6 pathways were involved in H8-EA.hy926 interaction under the LPA effect. Moreover, LPA increased the levels of IL-6 mRNA by cyclooxygenase-2 pathway in H8 cells. Collectively, LPA promotes trophoblast-endothelial crosstalk in vitro and induces the release of trophoblast soluble factors that stimulate endothelial cells migration without changes in apoptosis. The evidence presented here provides new insights about an active role of LPA as a lipid mediator regulating vascular remodeling at the maternal-fetal interface.
Asunto(s)
Células Endoteliales/efectos de los fármacos , Lisofosfolípidos/farmacología , Placentación/efectos de los fármacos , Placentación/fisiología , Trofoblastos/efectos de los fármacos , Línea Celular , Femenino , Humanos , Neovascularización Fisiológica/efectos de los fármacos , Neovascularización Fisiológica/fisiología , Embarazo , Receptor Cross-Talk/efectos de los fármacos , Receptor Cross-Talk/fisiología , Trofoblastos/metabolismoRESUMEN
The decidualization process involves phenotype and functional changes on endometrial cells and the modulation of mediators with immunoregulatory properties as the vasoactive intestinal peptide (VIP). We investigate VIP contribution to the decidualization program and to immunoregulation throughout the human embryo implantation process. The decidualization of Human endometrial stromal cell line (HESC) with Medroxyprogesterone-dibutyryl-cAMP increased VIP/VPAC-receptors system. In fact, VIP could induce decidualization increasing differentiation markers (IGFBP1, PRL, KLF13/KLF9 ratio, CXCL12, CXCL8 and CCL2) and allowing Blastocyst-like spheroids (BLS) invasion in an in vitro model of embryo implantation. Focus on the tolerogenic effects, decidualized cells induced a semi-mature profile on maternal dendritic cells; restrained CD4+ cells recruitment while increased regulatory T-cells recruitment. Interestingly, the human blastocyst conditioned media from developmentally impaired embryos diminished the invasion and T-regulatory cells recruitment in these settings. These evidences suggest that VIP contributes to the implantation process inducing decidualization, allowing BLS invasion and favoring a tolerogenic micro-environment.
Asunto(s)
Decidua/metabolismo , Implantación del Embrión/inmunología , Péptido Intestinal Vasoactivo/metabolismo , Biomarcadores/metabolismo , Blastocisto/citología , Línea Celular , Microambiente Celular/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Implantación del Embrión/efectos de los fármacos , Endometrio/citología , Femenino , Humanos , Tolerancia Inmunológica , Modelos Biológicos , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismo , Trofoblastos/efectos de los fármacos , Trofoblastos/metabolismoRESUMEN
Trophoblast infection by Toxoplasma gondii plays a pivotal role in the vertical transmission of toxoplasmosis. Here, we investigate whether the antibiotic therapy with azithromycin, spiramycin and sulfadiazine/pyrimethamine are effective to control trophoblast infection by two Brazilian T. gondii genotypes, TgChBrUD1 or TgChBrUD2. Two antibiotic protocols were evaluated, as follow: i) pre-treatment of T. gondii-tachyzoites with selected antibiotics prior trophoblast infection and ii) post-treatment of infected trophoblasts. The infection index/replication and the impact of the antibiotic therapy on the cytokine milieu were characterized. It was observed that TgChBrUD2 infection induced lower infection index/replication as compared to TgChBrUD1. Regardless the therapeutic protocol, azithromycin was more effective to control the trophoblast infection with both genotypes when compared to conventional antibiotics. Azithromycin induced higher IL-12 production in TgChBrUD1-infected cells that may synergize the anti-parasitic effect. In contrast, the effectiveness of azithromycin to control the TgChBrUD2-infection was not associated with the IL-12 production. BeWo-trophoblasts display distinct susceptibility to T. gondii genotypes and the azithromycin treatment showed to be more effective than conventional antibiotics to control the T. gondii infection/replication regardless the parasite genotype.
Asunto(s)
Antiprotozoarios/farmacología , Azitromicina/farmacología , Toxoplasma/efectos de los fármacos , Trofoblastos/parasitología , Línea Celular Tumoral , Citocinas/metabolismo , Combinación de Medicamentos , Genotipo , Humanos , Interleucina-12/metabolismo , Pirimetamina/farmacología , Espiramicina/farmacología , Sulfadiazina/farmacología , Toxoplasma/clasificación , Toxoplasma/genética , Toxoplasma/inmunología , Trofoblastos/efectos de los fármacosRESUMEN
Preeclampsia is a severe pregnancy complication globally, characterized by poor placentation triggering vascular dysfunction. Matrix metalloproteinases (MMPs) exhibit proteolytic activity implicated in the efficiency of trophoblast invasion to the uterine wall, and a dysregulation of these enzymes has been linked to preeclampsia. A decrease in MMP-2 and MMP-9 interferes with the normal remodeling of spiral arteries at early pregnancy stages, leading to the initial pathophysiological changes observed in preeclampsia. Later in pregnancy, an elevation in MMP-2 and MMP-9 induces abnormal release of vasoactive factors conditioning hypertension. Although these two enzymes lead the scene, other MMPs like MMP-1 and MMP-14 seem to have a role in this pathology. This review gathers published recent evidence about the implications of different MMPs in preeclampsia, and the potential use of these enzymes as emergent biomarkers and biological therapeutic targets, focusing on studies involving human subjects.
Asunto(s)
Metaloproteinasas de la Matriz/metabolismo , Preeclampsia/metabolismo , Adulto , Animales , Biomarcadores , Células Endoteliales/metabolismo , Femenino , Humanos , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Inhibidores de la Metaloproteinasa de la Matriz/uso terapéutico , Placenta/metabolismo , Placentación , Preeclampsia/tratamiento farmacológico , Preeclampsia/etiología , Embarazo , Trofoblastos/efectos de los fármacos , Trofoblastos/metabolismoRESUMEN
Placental barrier regulates maternal-fetal interchange protecting the baby from damage caused by substances found in the uterine environment or circulating in the vascular system. Organophosphate (OP) pesticides are a paramount group of environmental pollutants used in intensive agriculture for protection against diseases and pests. While many studies have reported an increased risk of pregnancy alterations in pregnant women exposed to OPs, few have analyzed the effects caused by these pesticides in the placenta. Herein, we evaluated the effects of chlorpyrifos (CPF), one of the most widely used OP insecticides, on human placenta using in vitro and ex vivo exposure models. Villous cytotrophoblast cells isolated from normal human term placentas maintained their cell viability, differentiated into syncytiotrophoblast-like structures, and increased the expression of ß-hCG, ABCG2, and P-gp in the presence of CPF at concentrations of 10 to 100µM. The same doses of CPF induced marked changes in chorionic villi samples. Indeed, CPF exposure increased stroma cell apoptosis, altered villi matrix composition, basement membrane thickness, and trophoblastic layer integrity. Histomorphological and ultrastructural alterations are compatible with those found in placentas where maternal-placenta injury is chronic and able to impair the placental barrier function and nutrient transport from mother to the fetus. Our study shows that placental ex vivo exposure to CPF produces tissue alterations and suggest that human placenta is a potential target of CPF toxicity. In addition, it highlights the importance of using different models to assess the effects of a toxic on human placenta.
Asunto(s)
Cloropirifos/toxicidad , Inhibidores de la Colinesterasa/toxicidad , Vellosidades Coriónicas/efectos de los fármacos , Insecticidas/toxicidad , Trofoblastos/efectos de los fármacos , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Apoptosis/efectos de los fármacos , Membrana Basal/efectos de los fármacos , Membrana Basal/ultraestructura , Bioensayo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Gonadotropina Coriónica Humana de Subunidad beta/metabolismo , Vellosidades Coriónicas/metabolismo , Vellosidades Coriónicas/ultraestructura , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Proteínas de Neoplasias/metabolismo , Embarazo , Reproducibilidad de los Resultados , Medición de Riesgo , Células del Estroma/efectos de los fármacos , Células del Estroma/ultraestructura , Factores de Tiempo , Técnicas de Cultivo de Tejidos , Pruebas de Toxicidad/métodos , Trofoblastos/metabolismo , Trofoblastos/ultraestructuraRESUMEN
PROBLEM: Women with antiphospholipid antibodies (aPLs) present a risk of pregnancy morbidity (PM), vascular thrombosis (VT), or both (PM/VT). aPLs affect trophoblast function, and the aim of this study was to determine the modulation of this aPL-induced damage by different drugs. METHOD OF STUDY: IgG was obtained from women with PM and PM/VT positive to aPLs. Binding of IgG to trophoblastic cells, proliferation, mitochondrial membrane integrity, and trophoblast invasion were assessed. The effect of enoxaparin, aspirin, and aspirin-triggered lipoxin (ATL) were evaluated as well as signal transducer and activator of transcription 3 (STAT3) phosphorylation. RESULTS: IgG from women with aPLs strongly binds to trophoblastic cells. Integrity of mitochondrial membrane was reduced, and proliferation was increased by IgG-PM/VT. Both IgG-PM and IgG-PM/VT decreased trophoblast invasion, which was restored by enoxaparin, aspirin, and ATL. IgG-PM triggered reduction in STAT3 phosphorylation. CONCLUSION: Some drugs used to prevent aPL-induced PM modulated the alteration of trophoblast function.
Asunto(s)
Anticuerpos Antifosfolípidos/inmunología , Síndrome Antifosfolípido/complicaciones , Complicaciones del Embarazo/inmunología , Trofoblastos/inmunología , Adulto , Anticoagulantes/farmacología , Síndrome Antifosfolípido/inmunología , Aspirina/farmacología , Western Blotting , Femenino , Citometría de Flujo , Humanos , Inmunoglobulina G/inmunología , Inmunohistoquímica , Lipoxinas/farmacología , Embarazo , Trofoblastos/efectos de los fármacos , Trofoblastos/patologíaRESUMEN
STUDY QUESTION: Do human trophoblast cells modulate neutrophil extracellular trap (NET) formation, reactive oxygen species (ROS) synthesis and neutrophil apoptosis through mechanisms involving vasoactive intestinal peptide (VIP)? SUMMARY ANSWER: Trophoblast cells inhibited NET formation and ROS synthesis and enhanced neutrophil apoptosis through VIP-mediated pathways in a model of maternal-placental interaction. WHAT IS KNOWN ALREADY: Immune homeostasis maintenance at the maternal-placental interface is mostly coordinated by trophoblast cells. Neutrophil activation and NET formation increases in pregnancies complicated by exacerbated pro-inflammatory responses. VIP has anti-inflammatory and immunosuppressant effects and is synthesized by trophoblast cells. STUDY DESIGN, SIZE, DURATION: This is a laboratory-based observational study that sampled circulating neutrophils from 50 healthy volunteers to explore their response in vitro to factors derived from human trophoblast cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: Peripheral blood neutrophils were isolated from healthy volunteers and tested in vitro with first trimester trophoblast cell line (Swan-71 and HTR8) conditioned media (CM) or with VIP. The effect of VIP and trophoblast CM on NET formation was assessed by co-localization of elastase and DNA by confocal microscopy, DNA release and elastase activity measurement. Neutrophil apoptosis was determined by flow cytometry or fluorescence microscopy. ROS formation was assessed by flow cytometry with a fluorescent probe. VIP silencing was performed by siRNA transfection. For phagocytosis of apoptotic neutrophils, autologous monocytes were sampled, and engulfment and cytokines were assessed by flow cytometry and ELISA. MAIN RESULTS AND THE ROLE OF CHANCE: Trophoblast CM and 10 nM VIP promoted neutrophil deactivation by preventing phorbol myristate acetate-induced NET formation and ROS synthesis while they increased neutrophil spontaneous apoptosis and reversed the anti-apoptotic effect of lipopolysaccharide (all P < 0.05 versus control). The effects of trophoblast CM were prevented by a VIP antagonist or when VIP knocked-down trophoblast cells were used (P < 0.05 versus control). Neutrophils driven to apoptosis by trophoblast CM could be rapidly engulfed by monocytes without increasing IL-12 production. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: The mechanisms of neutrophil deactivation by trophoblast VIP are based on the results obtained with neutrophils drawn from peripheral blood of healthy individuals interacting with trophoblast cell lines in vitro. These studies were designed to investigate biological processes at the cellular and molecular level; therefore, they have the limitations of studies in vitro and it is not possible to ascertain if these mechanisms operate similarly in vivo. We tested 50 neutrophil samples from healthy volunteers that have a normal variability in their responses. Cell lines derived from human trophoblast were used, and we cannot rule out a differential behavior of trophoblast cells in contact with neutrophils in vivo. WIDER IMPLICATIONS OF THE FINDINGS: Results presented here are consistent with an active mechanism through which neutrophils in contact with trophoblast cells would be deactivated and silently cleared by decidual macrophages throughout pregnancy. They support a novel immunomodulatory role of trophoblast VIP on neutrophils at the placenta, providing new clues for pharmacological targeting of immune and trophoblast cells in pregnancy complications associated with exacerbated inflammation. STUDY FUNDING/COMPETING INTERESTS: This work was funded by the National Agency of Sciences and Technology (PICT 2011-0144, 2014-0657 and 2013-2177) and University of Buenos Aires (UBACyT 20020130100040BA, 20020150100161BA and 20020130100744BA). The authors declare no competing interests.
Asunto(s)
Apoptosis/fisiología , Trampas Extracelulares/metabolismo , Transducción de Señal/fisiología , Trofoblastos/metabolismo , Péptido Intestinal Vasoactivo/farmacología , Apoptosis/efectos de los fármacos , Línea Celular , Medios de Cultivo Condicionados/farmacología , Trampas Extracelulares/efectos de los fármacos , Femenino , Humanos , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Fagocitosis/efectos de los fármacos , Fagocitosis/fisiología , Embarazo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Trofoblastos/efectos de los fármacosRESUMEN
Lipopolysaccharide (LPS) injections during pregnancy are well established as models for pregnancy complications, including fetal growth restriction (FGR), thrombophilia, preterm labor and abortion. Indeed, inflammation, as induced by LPS injection has been described as a pivotal factor in cases of miscarriage related to placental tissue damage. The phosphodiesterase-5 inhibitor sildenafil (Viagra®) is currently used to treat FGR cases in women, while low-molecular weight heparin (Fragmin®) is a standard treatment for recurrent miscarriage (RM). However, the pathways and cellular dynamics involved in RM are not completely understood. The aim of this study was to evaluate the protective effect of sildenafil and dalteparin in a mouse model of LPS-induced abortion. Histopathology, ultrastructural analysis and immunofluorescence for P-selectin were studied in two different placental cell types: trophoblast cells and labyrinth endothelial cells. Treatment with sildenafil either alone or in combination with heparin showed the best response against LPS-induced injury during pregnancy. In conclusion, our results support the use of these drugs as future therapeutic agents that may protect the placenta against inflammatory injury in RM events. Analyses of the ultrastructure and placental immunophysiology are important to understand the mechanism underlying RM. These findings may spark future studies and aid in the development of new therapies in cases of RM.