Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.581
Filtrar
1.
Bratisl Lek Listy ; 121(3): 225-229, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32115981

RESUMO

AIM: Nicotine at high concentrations induces apoptosis in trophoblastic cells through induction of cell cytotoxicity and Reactive Oxygen Species (ROS). Methamphetamine in low dose has pharmaceutical properties. It seems that this components in low dose can protect the trophoblastic cells from nicotine-induced cell death. METHOD: Trophoblastic (JEG-3) cells grown in DMEM culture medium. MTT assay test detected the cell viability and Lactate Dehydrogenase test measured the cells cytotoxicity. Griess reaction was used for NO production analysis. Cell migration traced by wounding technique. Human Cytokine Array Focused 13-plex was also used for analysis of IL-1α, IL-1ß, IL-6, INFγ, and TNFα pre-inflammatory cytokines. RESULTS: Methamphetamine, in very low dose (pM), increased the cell viability and NO production, and decreased cell cytotoxicity, IL-1α, IL-1ß, IL-6, INFγ, and TNFα pre-inflammatory cytokines of JEG-3 cell which were exposed to high dose of nicotine, respectively. Cell migration was enhanced by low dose of methamphetamine in JEG-3 cells. CONCLUSION: Methamphetamine in very low dose suppressed the JEG-3 cell death induced by high dose of nicotine (Fig. 5, Ref. 48) Keywords: methamphetamine, nicotine, cell death, NO.


Assuntos
Dopaminérgicos , Inflamação , Metanfetamina , Trofoblastos , Linhagem Celular Tumoral , Sobrevivência Celular , Dopaminérgicos/farmacologia , Humanos , Inflamação/tratamento farmacológico , Metanfetamina/farmacologia , Nicotina/toxicidade , Trofoblastos/efeitos dos fármacos
2.
PLoS One ; 15(2): e0229332, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32092105

RESUMO

The placenta, a tissue that is metabolically active and rich in mitochondria, forms a critical interface between the mother and developing fetus. Oxidative stress within this tissue, derived from the dysregulation of reactive oxygen species (ROS), has been linked to a number of adverse fetal outcomes. While such outcomes have been associated with mitochondrial dysfunction, the causal role of mitochondrial dysfunction and mitochondrially generated ROS in altering the process of placentation remains unclear. In this study, mitochondrial complex I activity was attenuated using 10 nM rotenone to induce cellular oxidative stress by increasing mitochondrial ROS production in the BeWo choriocarcinoma cell line. Increased mitochondrial ROS resulted in a significant decrease in the transcripts which encode for proteins associated with fusion (GCM1, ERVW-1, and ERVFRD-1) resulting in a 5-fold decrease in the percentage of BeWo fusion. This outcome was associated with increased indicators of mitochondrial fragmentation, as determined by decreased expression of MFN2 and OPA1 along with an increase in a marker of mitochondrial fission (DRP1). Importantly, increased mitochondrial ROS also resulted in a 5.0-fold reduction of human placental lactogen (PL) and a 4.4-fold reduction of insulin like growth factor 2 (IGF2) transcripts; hormones which play an important role in regulating fetal growth. The pre-treatment of rotenone-exposed cells with 5 mM N-acetyl cysteine (NAC) resulted in the prevention of these ROS mediated changes in BeWo function and supports a central role for mitochondrial ROS signaling in the maintenance and function of the materno-fetal interface.


Assuntos
Mitocôndrias/metabolismo , Hormônios Placentários/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Trofoblastos/metabolismo , Fusão Celular , Células Cultivadas , Feminino , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Gravidez , Espécies Reativas de Oxigênio/farmacologia , Rotenona/farmacologia , Transdução de Sinais/efeitos dos fármacos , Trofoblastos/efeitos dos fármacos
3.
Am J Pathol ; 190(2): 388-399, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31955792

RESUMO

Preterm premature rupture of membranes (PPROM) and thrombin generation by decidual cell-expressed tissue factor often accompany abruptions. Underlying mechanisms remain unclear. We hypothesized that thrombin-induced colony-stimulating factor-2 (CSF-2) in decidual cells triggers paracrine signaling via its receptor (CSF2R) in trophoblasts, promoting fetal membrane weakening and abruption-associated PPROM. Decidua basalis sections from term (n = 10), idiopathic preterm birth (PTB; n = 8), and abruption-complicated pregnancies (n = 8) were immunostained for CSF-2. Real-time quantitative PCR measured CSF2 and CSF2R mRNA levels. Term decidual cell (TDC) monolayers were treated with 10-8 mol/L estradiol ± 10-7 mol/L medroxyprogesterone acetate (MPA) ± 1 IU/mL thrombin pretreatment for 4 hours, washed, and then incubated in control medium with estradiol ± MPA. TDC-derived conditioned media supernatant effects on fetal membrane weakening were analyzed. Immunostaining localized CSF-2 primarily to decidual cell cytoplasm and cytotrophoblast cell membranes. CSF-2 immunoreactivity was higher in abruption-complicated or idiopathic PTB specimens versus normal term specimens (P < 0.001). CSF2 mRNA was higher in TDCs versus cytotrophoblasts (P < 0.05), whereas CSF2R mRNA was 1.3 × 104-fold higher in cytotrophoblasts versus TDCs (P < 0.001). Thrombin enhanced CSF-2 secretion in TDC cultures fourfold (P < 0.05); MPA reduced this effect. Thrombin-pretreated TDC-derived conditioned media supernatant weakened fetal membranes (P < 0.05), which MPA inhibited. TDC-derived CSF-2, acting via trophoblast-expressed CSFR2, contributes to thrombin-induced fetal membrane weakening, eliciting abruption-related PPROM and PTB.


Assuntos
Descolamento Prematuro da Placenta/fisiopatologia , Decídua/patologia , Membranas Extraembrionárias/patologia , Ruptura Prematura de Membranas Fetais/patologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Nascimento Prematuro/etiologia , Trombina/farmacologia , Decídua/efeitos dos fármacos , Decídua/metabolismo , Membranas Extraembrionárias/metabolismo , Feminino , Ruptura Prematura de Membranas Fetais/etiologia , Ruptura Prematura de Membranas Fetais/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Humanos , Gravidez , Nascimento Prematuro/metabolismo , Nascimento Prematuro/patologia , Transdução de Sinais , Trofoblastos/efeitos dos fármacos , Trofoblastos/metabolismo , Trofoblastos/patologia
4.
Toxicol Lett ; 319: 129-137, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31730886

RESUMO

The increasing use of synthetic cannabinoids (SCBs) in recreational settings is becoming a new paradigm of drug abuse. Although SCBs effects mimic those of the Cannabis sativa plant, these drugs are frequently more potent and hazardous. It is known that endocannabinoid signalling plays a crucial role in diverse reproductive events such as placental development. Moreover, the negative impact of the phytocannabinoid Δ9-tetrahydrocannabinol (THC) in pregnancy outcome, leading to prematurity, intrauterine growth restriction and low birth weight is well recognized, which makes women of childbearing age a sensitive group to developmental adverse effects of cannabinoids. Placental trophoblast turnover relies on regulated processes of proliferation and apoptosis for normal placental development. Here, we explored the impact of the SCBs JWH-018, JWH-122 and UR-144 and of the phytocannabinoid THC in BeWo cell line, a human placental cytotrophoblast cell model. All the cannabinoids caused a significant decrease in cell viability without LDH release, though this effect was only detected for the highest concentrations of THC. Moreover, a cell cycle arrest at the G2/M phase was also observed. JWH-018 and JWH-122 increased reactive oxygen species (ROS) production and THC, UR-144 and JWH-122 caused loss of mitochondrial membrane potential. All the compounds were able to induce caspase-9 activation. The involvement of apoptotic pathways was further confirmed through the significant increase in caspase -3/-7 activities. For UR-144, this effect was reversed by the CB1 antagonist AM281, for JWH-018 and THC this effect was mediated by both cannabinoid receptors CB1 and CB2 while for JWH-122 it was cannabinoid receptor-independent. This work demonstrates that THC and SCBs are able to induce apoptotic cell death. Although they may act through different mechanisms and potencies, the studied cannabinoids have the potential to disrupt gestational fundamental events.


Assuntos
Apoptose/efeitos dos fármacos , Canabinoides/toxicidade , Dronabinol/toxicidade , Indóis/toxicidade , Naftalenos/toxicidade , Placenta/citologia , Placenta/efeitos dos fármacos , Adulto , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Feminino , Humanos , L-Lactato Desidrogenase/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Gravidez , Espécies Reativas de Oxigênio/metabolismo , Trofoblastos/efeitos dos fármacos
5.
J Steroid Biochem Mol Biol ; 195: 105470, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31509772

RESUMO

Serotonin reuptake inhibitors (SRIs) are currently the main molecules prescribed to pregnant women that suffer from depression. Placental cells are exposed to SRIs via maternal blood, and we have previously shown that SRIs alter feto-placental steroidogenesis in an in vitro co-culture model. More specifically, serotonin (5-HT) regulates the estrogen biosynthetic enzyme aromatase (cytochrome P450 19; CYP19), which is disrupted by fluoxetine and its active metabolite norfluoxetine in BeWo choriocarcinoma cells. Based on molecular simulations, the present study illustrates that the SRIs fluoxetine, norfluoxetine, paroxetine, sertraline, citalopram and venlafaxine exhibit binding affinity for the active-site pocket of CYP19, suggesting potential competitive inhibition. Using BeWo cells and primary villous trophoblast cells isolated from normal term placentas, we compared the effects of the SRIs on CYP19 activity. We observed that paroxetine and sertraline induce aromatase activity in BeWo cells, while venlafaxine, fluoxetine, paroxetine and sertraline decrease aromatase activity in primary villous trophoblast. The effects of paroxetine and sertraline in primary villous trophoblasts were observed at the lower doses tested. We also showed that 5-HT and the 5-HT2A receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) induced CYP19 activity. An increase in phosphorylation of serine and tyrosine and a decrease in threonine phosphorylation of CYP19 was also associated with DOI treatment. Our results contribute to better understanding how 5-HT and SRIs interact with CYP19 and may affect estrogen production. Moreover, this study suggests that alteration of placental 5-HT levels due to depression and/or SRI treatment during pregnancy may be associated with disruption of placental estrogen production.


Assuntos
Aromatase/metabolismo , Placenta/efeitos dos fármacos , Inibidores de Captação de Serotonina/farmacologia , Serotonina/farmacologia , Células Cultivadas , Citalopram/farmacologia , Feminino , Fluoxetina/análogos & derivados , Fluoxetina/farmacologia , Humanos , Simulação de Acoplamento Molecular , Paroxetina/farmacologia , Placenta/metabolismo , Gravidez , Sertralina/farmacologia , Trofoblastos/efeitos dos fármacos , Trofoblastos/metabolismo , Cloridrato de Venlafaxina/farmacologia
6.
Int J Mol Sci ; 20(18)2019 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-31500240

RESUMO

Omega-3 fatty acids are important to pregnancy and neonatal development and health. One mechanism by which omega-3 fatty acids exert their protective effects is through serving as substrates for the generation of specialized pro-resolving lipid mediators (SPM) that potently limit and resolve inflammatory processes. We recently identified that SPM levels are increased in maternal blood at delivery as compared to umbilical cord blood, suggesting the placenta as a potential site of action for maternal SPM. To explore this hypothesis, we obtained human placental samples and stained for the SPM resolvin D2 (RvD2) receptor GPR18 via immunohistochemistry. In so doing, we identified GPR18 expression in placental vascular smooth muscle and extravillous trophoblasts of the placental tissues. Using in vitro culturing, we confirmed expression of GPR18 in these cell types and further identified that stimulation with RvD2 led to significantly altered responsiveness (cytoskeletal changes and pro-inflammatory cytokine production) to lipopolysaccharide inflammatory stimulation in human umbilical artery smooth muscle cells and placental trophoblasts. Taken together, these findings establish a role for SPM actions in human placental tissue.


Assuntos
Ácidos Docosa-Hexaenoicos/farmacologia , Músculo Liso Vascular/citologia , Receptores Acoplados a Proteínas-G/genética , Trofoblastos/citologia , Adulto , Células Cultivadas , Ácidos Graxos Ômega-3/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Idade Materna , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Placenta/citologia , Placenta/efeitos dos fármacos , Placenta/metabolismo , Gravidez , Receptores Acoplados a Proteínas-G/metabolismo , Trofoblastos/efeitos dos fármacos , Trofoblastos/metabolismo , Adulto Jovem
7.
Environ Pollut ; 254(Pt A): 112991, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31421572

RESUMO

Cadmium (Cd), a ubiquitous environmental pollutant, is known to impair placental development. However, the underlying mechanisms remain unclear. The present study used in vivo and in vitro models to investigate the effects of Cd on apoptosis and autophagy in placental trophoblasts and its mechanism. Pregnant mice were exposed to CdCl2 (4.5 mg/kg) on gestational day (GD) 9. Human JEG-3 cells were exposed to CdCl2 (0-40 µM) for different time points. Gestational Cd exposure obviously lowered the weight and diameter of mouse placentas. Number of TUNEL-positive cells was markedly elevated in Cd-administered mouse placentas and JEG-3 cells. Correspondingly, Cd significantly up-regulated cleaved caspase-3 protein level, a key indicator of apoptosis, in murine placentas and JEG-3 cells. Simultaneously, Cd also triggered autophagy, as determined by an elevation of LC3B-II and p62 protein, and accumulation of LC3-positive puncta, in placental trophoblasts. Chloroquine an autophagy inhibitor, obviously aggravated Cd-induced apoptosis in JEG-3 cells. By contrast, rapamycin, a specific autophagy inducer, significantly alleviated Cd-triggered apoptosis in JEG-3 cells. Mechanistically, autophagy inhibited Cd-induced apoptosis mainly via degrading caspase-9. Co-localizations of p62, a classical autophagic receptor, and caspase-9 were observed in Cd-stimulated human JEG-3 cells. Moreover, p62 siRNAs pretreatment markedly blocked the degradation of caspase 9 proteins via Cd-activated autophagy in JEG-3 cells. Collectively, our data suggest that activation of autophagy inhibits Cd-induced apoptosis via p62-mediated caspase-9 degradation in placental trophoblasts. These findings provide a new mechanistic insight into Cd-induced impairments of placental and fetal development.


Assuntos
Autofagia/fisiologia , Cádmio/toxicidade , Substâncias Perigosas/toxicidade , Placenta/fisiologia , Trofoblastos/fisiologia , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Cádmio/metabolismo , Caspase 3 , Caspase 9 , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Placenta/metabolismo , Gravidez , Testes de Toxicidade , Trofoblastos/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
8.
Pestic Biochem Physiol ; 159: 144-153, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31400776

RESUMO

Ivermectin is a pesticide that has been used for over 30 years in livestock. Although there are a number of studies on the therapeutic potential of ivermectin, little is known about the effects of the drug during the early stage of pregnancy. In this study, we investigated the detrimental effects of ivermectin on porcine trophectoderm (pTr) and uterine luminal epithelial (pLE) cells. Ivermectin not only inhibited the proliferation of both cells via the regulation of cell cycle-associated genes, but also induced apoptosis in pTr and pLE cells. We also verified its effect on mitochondrial dysfunction as shown by loss of mitochondrial membrane potential, mitochondrial Ca2+ overload, and reactive oxygen species (ROS) generation in pTr and pLE cells. As a mechanistic approach, we evaluated ivermectin-mediated cell signaling interactions including PI3K, AKT and MAPK pathways. Overall, our results suggest that constant exposure to and accumulation of ivermectin may cause abnormal fetal morphogenesis and placentation during the early stages of pregnancy. Our results may further provide a comprehensive understanding of the detrimental effects of ivermectin during pregnancy and will contribute to the establishment of a complete safety profile for ivermectin and its association with environmental pollution and public health in humans and livestock.


Assuntos
Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Ivermectina/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Útero/citologia , Animais , Proliferação de Células/efeitos dos fármacos , Feminino , Suínos , Trofoblastos/citologia , Trofoblastos/efeitos dos fármacos
9.
PLoS One ; 14(7): e0218799, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31318865

RESUMO

Ambient air pollution is considered a major environmental health threat to pregnant women. Our previous work has shown an association between exposure to airborne particulate matter (PM) and an increased risk of developing pre-eclamspia. It is now recognized that many pregnancy complications are due to underlying placental dysfunction, and this tissue plays a pivotal role in pre-eclamspia. Recent studies have shown that PM can enter the circulation and reach the human placenta but the effects of PM on human placental function are still largely unknown. In this work we investigated the effects of airborne PM on trophoblast cells. Human, first trimester trophoblast cells (HTR-8/SV) were exposed to urban pollution particles (Malmö PM2.5; Prague PM10) for up to seven days in vitro and were analysed for uptake, levels of hCGß and IL-6 secretion and proteomic analysis. HTR-8/SVneo cells rapidly endocytose PM within 30 min of exposure and particles accumulate in the cell in perinuclear vesicles. High doses of Prague and Malmö PM (500-5000 ng/ml) significantly decreased hCGß secretion and increased IL-6 secretion after 48 h exposure. Exposure to PM (50 ng/ml) for 48h or seven days led to reduced cellular growth and altered protein expression. The differentially expressed proteins are involved in networks that regulate cellular processes such as inflammation, endoplasmic reticulum stress, cellular survival and molecular transport pathways. Our studies suggest that trophoblast cells exposed to low levels of urban PM respond with reduced growth, oxidative stress, inflammation and endoplasmic reticulum stress after taking up the particles by endocytosis. Many of the dysfunctional cellular processes ascribed to the differentially expressed proteins in this study, are similar to those described in PE, suggesting that low levels of urban PM may disrupt cellular processes in trophoblast cells. Many of the differentially expressed proteins identified in this study are involved in inflammation and may be potential biomarkers for PE.


Assuntos
Poluição do Ar/efeitos adversos , Inflamação/genética , Pré-Eclâmpsia/genética , Trofoblastos/efeitos dos fármacos , Poluentes Atmosféricos/efeitos adversos , Sobrevivência Celular/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/induzido quimicamente , Inflamação/patologia , Interleucina-6/genética , Estresse Oxidativo/efeitos dos fármacos , Material Particulado/efeitos adversos , Placenta/efeitos dos fármacos , Placenta/patologia , Pré-Eclâmpsia/induzido quimicamente , Pré-Eclâmpsia/patologia , Gravidez , Complicações na Gravidez/induzido quimicamente , Complicações na Gravidez/genética , Complicações na Gravidez/patologia , Proteômica/métodos , Trofoblastos/metabolismo , Trofoblastos/patologia
10.
Mol Med Rep ; 20(2): 1451-1458, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31173227

RESUMO

Preeclampsia (PE) is a complication of pregnancy, and a leading cause of maternal mortality and morbidity worldwide. Recently, the dysregulation of long non­coding RNAs (lncRNAs) has been reported to contribute to the pathogenesis and progression of PE. This study aimed to examine the alterations in the lncRNA family with sequence similarity 99 member A (FAM99A) in PE and its effects on trophoblasts. The results of reverse transcription­quantitative PCR indicated that the expression levels of FAM99A were downregulated in placental tissues from women with severe PE compared with in those from controls. A Transwell invasion assay and wound healing assay revealed that overexpression of FAM99A promoted invasion and migration of HTR­8/SVneo cells; conversely, knockdown of FAM99A suppressed the invasive and migratory abilities of HTR­8/SVneo cells. Flow cytometry demonstrated that FAM99A overexpression induced a decrease in the apoptotic rate of cells, whereas knockdown of FAM99A increased the apoptotic rate of HTR­8/SVneo cells. Western blot analysis revealed that overexpression of FAM99A decreased the protein expression levels of cleaved caspase­3, cleaved caspase­9 and Bax, and increased Bcl­2 protein expression, whereas knockdown of FAM99A had the opposite effects on these protein levels. Overexpression of FAM99A also decreased caspase­3 activity in HTR­8/SVneo cells; however, knockdown of FAM99A increased caspase­3 activity. In addition, overexpression of FAM99A enhanced Wnt/ß­catenin signaling activity, whereas FAM99A knockdown exerted an inhibitory effect on the Wnt/ß­catenin signaling activity in HTR­8/SVneo cells. In conclusion, these results indicated that FAM99A may serve a role in modulating the functions of trophoblasts, partially via targeting Wnt/ß­catenin signaling.


Assuntos
Pré-Eclâmpsia/genética , RNA Longo não Codificante/genética , Trofoblastos/metabolismo , Via de Sinalização Wnt/genética , Adulto , Apoptose/efeitos dos fármacos , Estudos de Casos e Controles , Caspase 3/genética , Caspase 3/metabolismo , Caspase 9/genética , Caspase 9/metabolismo , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Pré-Eclâmpsia/metabolismo , Pré-Eclâmpsia/patologia , Gravidez , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Trofoblastos/efeitos dos fármacos , Trofoblastos/patologia , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
11.
Zygote ; 27(4): 255-258, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31218974

RESUMO

This study aimed to optimize the derivation of trophectoderm from in vitro-produced camel embryos under feeder-free culture conditions using the basement membrane matrix Matrigel. Trophoblastic vesicles were obtained through mechanical microdissection of in vitro-produced camel (Camelus dromedarius) embryos. Supplementing the culture medium with 10 ng/ml of epidermal growth factor and 10 ng/ml fibroblast growth factor improved the attachment and subsequent outgrowths of cultured trophoblastic vesicles when compared with the control group and the groups supplemented individually with each growth factor. The expression levels of pluripotency genes octamer-binding transcription factor 4 (Oct4), sex determining region Y-box 2 (Sox2), myelocytomatosis proto-oncogene (c-Myc) and anti-apoptotic gene B-cell lymphoma 2 (Bcl2) were increased in trophoblastic vesicles supplemented with both growth factors when compared with the control group. Conversely, both growth factors decreased the expression of apoptotic genes tumour protein p53 (p53) and Bcl-2-associated X protein (Bax). To the best of our knowledge, this may be the first report describing the derivation of trophoblast stem cells from in vitro-produced camel embryos.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Embrião de Mamíferos/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Trofoblastos/efeitos dos fármacos , Animais , Camelus , Diferenciação Celular/genética , Células Cultivadas , Sinergismo Farmacológico , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Fator 3 de Transcrição de Octâmero/genética , Proteínas Proto-Oncogênicas c-myb/genética , Fatores de Transcrição SOXB1/genética , Trofoblastos/citologia , Trofoblastos/metabolismo
12.
Biomed Pharmacother ; 117: 109125, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31226636

RESUMO

Titanium dioxide nanoparticles (TiO2 NPs) are used extensively in our daily lives, and their toxic effects on the placenta have been reported. Animal studies indicated that placental development is impaired after maternal exposure of TiO2 NPs, but the underlying mechanisms remain largely unknown. In the present study, we used a human trophoblast-derived cell, HTR8-SVneo, to determine how TiO2 NPs affected placental functions, and found out potential reversal targets. TEM was employed for TiO2 NPs morphology observation and uptake assessment. RT-PCR was used to detect the expression of both mRNA and miRNA, and western blotting was used for protein examination. Cell invasion ability was evaluated by Transwell assay, and cytoskeletons were observed by immunofluorescence combined with confocal microscope examination. We found that TiO2 NPs disrupted cytoskeletons and impaired cell invasion ability. Further investigations showed that TiO2 NPs increased the expression of a microRNA (miR-96-5p), which targeted and down-regulated the translation of EZR mRNA, a gene that encodes ezrin protein, and affected the cell cytoskeletons and ultimately cell invasion ability. When the expression of miR-96-5p was down-regulated, the expression level of ezrin protein was also reversed, and cell invasion ability was partially restored. Collectively, we determined how miR-96-5p mediates TiO2 NP-induced placental dysfunction, and provided a potential rescue target for future therapy.


Assuntos
Movimento Celular/efeitos dos fármacos , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Nanopartículas Metálicas/química , MicroRNAs/genética , Titânio/farmacologia , Trofoblastos/patologia , Regulação para Cima , Linhagem Celular , Proteínas do Citoesqueleto/genética , Citoesqueleto/efeitos dos fármacos , Humanos , Nanopartículas Metálicas/ultraestrutura , MicroRNAs/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Trofoblastos/efeitos dos fármacos , Trofoblastos/metabolismo , Regulação para Cima/efeitos dos fármacos
13.
Endocrinology ; 160(6): 1492-1505, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31002314

RESUMO

Extravillous trophoblast (EVT) uterine artery remodeling (UAR) promotes placental blood flow, but UAR regulation is unproven. Elevating estradiol (E2) in early baboon pregnancy suppressed UAR and EVT vascular endothelial growth factor (VEGF) expression, but this did not prove that VEGF mediated this process. Therefore, our primate model of prematurely elevating E2 and contrast-enhanced ultrasound cavitation of microbubble (MB) carriers was used to deliver VEGF DNA to the placental basal plate (PBP) to establish the role of VEGF in UAR. Baboons were treated on days 25 to 59 of gestation (term, 184 days) with E2 alone or with E2 plus VEGF DNA-conjugated MBs briefly infused via a maternal peripheral vein on days 25, 35, 45, and 55. At each of these times an ultrasound beam was directed to the PBP to collapse the MBs and release VEGF DNA. VEGF DNA-labeled MBs per contrast agent was localized in the PBP but not the fetus. Remodeling of uterine arteries >25 µm in diameter on day 60 was 75% lower (P < 0.001) in E2-treated (7% ± 2%) than in untreated baboons (30% ± 4%) and was restored to normal by E2/VEGF. VEGF protein levels (signals/nuclear area) within the PBP were twofold lower (P < 0.01) in E2-treated (4.2 ± 0.9) than in untreated (9.8 ± 2.8) baboons and restored to normal by E2/VEGF (11.9 ± 1.6), substantiating VEGF transfection. Thus, VEGF gene delivery selectively to the PBP prevented the decrease in UAR elicited by prematurely elevating E2 levels, establishing the role of VEGF in regulating UAR in vivo during primate pregnancy.


Assuntos
Estradiol/farmacologia , Placenta/efeitos dos fármacos , Artéria Uterina/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/farmacologia , Remodelação Vascular/efeitos dos fármacos , Animais , Feminino , Papio , Placenta/metabolismo , Gravidez , Trofoblastos/efeitos dos fármacos , Trofoblastos/metabolismo
14.
Biomed Pharmacother ; 116: 108836, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31004838

RESUMO

BACKGROUND: An impaired trophoblast invasion ability contributes to the development of pre-eclampsia (PE), and can be induced by the altered expression of various microRNAs (miRs). MiR-141 and CXCL12ß (C-X-C motif chemokine ligand 12) signaling regulate trophoblast invasion and vascularization capabilities during PE pathogenesis; however, their interactions and underlying mechanisms of action remain unclear. We investigated how miR-141 modulates trophoblast invasion, with a focus on its interaction with CXCL12ß signaling. METHODS: A PE model was established by using HTR-8/SVneo cells, which were first cultured with 2% O2 for 48 h, and then with 5% O2. The expression of miR-141 in human villous trophoblast HTR-8/SVneo cells was modulated with mimics or an inhibitor, and analyzed by quantitative RT-PCR. CXCL12ß levels were determined by ELISA. Cell apoptosis was determined by flow cytometry, and the invasion and vascularization capabilities of trophoblasts were evaluated by Transwell and tube formation assays, respectively. Binding of miR-141 with CXCL12ß mRNA was verified by the dual luciferase assay. Protein levels were estimated by western blotting. RESULTS: MiR-141 expression was significantly induced by hypoxia in HTR-8/SVneo cells. MiR-141 was found to promote apoptosis and inhibit the invasion and vascularization abilities of HTR-8/SVneo cells under conditions of hypoxia. MiR-141 could directly bind with the 3'UTR region of CXCL12ß mRNA and inhibit its translation. In addition, we proved that miR-141 could inhibit the invasion and vascularization abilities, and promote the apoptosis of HTR-8/SVneo cells by targeting CXCL12ß under hypoxic conditions. Furthermore, we demonstrated that arachidonic acid could reverse the invasion and apoptosis abilities of HTR-8/SVneo cells mediated by CXCL12ß during hypoxia. In terms of mechanism, MiR-141 could downregulate MMP2, p62, and LC3B expression, and upregulate ROCK1 and RhoA expression in HTR-8/SVneo cells by targeting the CXCL12ß gene during hypoxia. The effects of CXCL12ßon HTR-8/SVneo cells could be reversed by arachidonic acid (ARA). CONCLUSION: Induction of miR-141 by hypoxia promotes apoptosis, and inhibits the invasion and vascularization capabilities of HTR-8/SVneo cells by suppressing the CXCL12ß and CXCR2/4 signaling pathways.


Assuntos
Apoptose , Quimiocina CXCL12/metabolismo , MicroRNAs/metabolismo , Neovascularização Fisiológica , Receptores CXCR4/metabolismo , Receptores de Interleucina-8B/metabolismo , Transdução de Sinais , Trofoblastos/patologia , Apoptose/efeitos dos fármacos , Ácido Araquidônico/farmacologia , Sequência de Bases , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , MicroRNAs/genética , Modelos Biológicos , Neovascularização Fisiológica/efeitos dos fármacos , Trofoblastos/efeitos dos fármacos , Trofoblastos/metabolismo
15.
Biochem Pharmacol ; 164: 289-298, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31022396

RESUMO

Adequate invasion and complete remodelling of the maternal spiral arteries by the invading extravillous trophoblasts are the major determinants of a successful pregnancy. Increase in oxidative stress during pregnancy has been linked to the reduction in trophoblast invasion and incomplete conversion of the maternal spiral arteries, resulting in pregnancy complications such as pre-eclampsia, intrauterine growth restriction, and spontaneous miscarriages resulting in foetal/maternal mortality. The use of antioxidant therapy (vitamin C and E) and other preventative treatments (such as low dose aspirin) have been ineffective in preventing pre-eclampsia. Also, as the majority of antihypertensive drugs pose side effects, choosing an appropriate treatment would depend upon the efficacy and safety of mother/foetus. Since pre-eclampsia is mainly linked to placental oxidative stress, new diet-based antioxidants can be of use to prevent this condition. The antioxidant properties of flavonoids (naturally occurring phenolic compounds which are ubiquitously distributed in fruits and vegetables) have been well documented in non-trophoblast cells. Therefore, this study aimed to investigate the effects of flavonoids (quercetin, hesperidin) and their metabolites (Quercetin 3-O-ß-glucuronide and hesperetin), either alone or in combination, on first trimester trophoblast cell line HTR-8/SVneo during oxidative stress. The data obtained from this study indicate that selected flavonoids, their respective metabolites significantly reduced the levels of reduced glutathione (p < 0.0001) during HR-induced oxidative stress. These flavonoids also inhibited the activation of pro-apoptotic kinases (p38 MAPK and c-Jun N-terminal kinase) during HR-induced phosphorylation. In addition, they enhanced spheroid stem-like cell generation from HTR8/SVneo cells, aiding their invasion. Our data suggest that dietary intake of food rich in quercetin or hesperidin during early pregnancy can significantly improve trophoblast (placenta) health and function against oxidative stress.


Assuntos
Autorrenovação Celular/efeitos dos fármacos , Flavonoides/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Compostos Fitoquímicos/farmacologia , Primeiro Trimestre da Gravidez/efeitos dos fármacos , Trofoblastos/efeitos dos fármacos , Linhagem Celular , Autorrenovação Celular/fisiologia , Citoproteção/efeitos dos fármacos , Citoproteção/fisiologia , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Feminino , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Estresse Oxidativo/fisiologia , Gravidez , Primeiro Trimestre da Gravidez/metabolismo , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Trofoblastos/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
16.
Am J Physiol Regul Integr Comp Physiol ; 316(5): R628-R639, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30892908

RESUMO

We investigated expression of cholecystokinin (CCK) in humans and mice, and the bitter taste receptor TAS2R14 in the human placenta. Because CCK and gastrin activate the CCKBR receptor, we also explored placental gastrin expression. Finally, we investigated calcium signaling by CCK and TAS2R14. By RT-PCR, we found CCK/Cck and GAST/Gast mRNA expression in both normal human and mouse placentas, as well as in human trophoblast cell lines (TCL). Although both Cckar and -br mRNA were expressed in the mouse placenta, only CCKBR mRNA was detected in the human placenta and TCL. mRNA expression for TAS2R14 was also observed in the human placenta and TCL. Using immunohistochemistry, CCK protein was localized to the syncytiotrophoblast (ST) and extravillous trophoblast (EVT) in the human term placenta, and to trophoblast glycogen cells in mouse and human placentas. Gastrin and TAS2R14 proteins were also observed in ST and EVT of the human placenta. Both sulfated and nonsulfated CCK elicited a comparable rise in intracellular calcium in TCL, consistent with CCKBR expression. Three TAS2R14 agonists, flufenamic acid, chlorhexidine, and diphenhydramine, also evoked rises in intracellular calcium in TCL. These results establish CCK, gastrin, and their receptor(s) in both human and mouse placentas, and TAS2R14 in the human placenta. Both CCK and TAS2R14 agonists increased intracellular calcium in human TCL. Although the roles of these ligands and receptors, and their potential cross talk in normal and pathological placentas, are currently unknown, this study opens new avenues for placental research.


Assuntos
Colecistocinina/metabolismo , Gastrinas/metabolismo , Receptor de Colecistocinina B/metabolismo , Receptores da Colecistocinina/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais , Trofoblastos/metabolismo , Animais , Sinalização do Cálcio , Linhagem Celular , Colecistocinina/genética , Colecistocinina/farmacologia , Feminino , Gastrinas/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor de Colecistocinina B/genética , Receptores da Colecistocinina/agonistas , Receptores da Colecistocinina/genética , Receptores Acoplados a Proteínas-G/agonistas , Receptores Acoplados a Proteínas-G/genética , Transdução de Sinais/efeitos dos fármacos , Trofoblastos/efeitos dos fármacos
17.
Am J Physiol Endocrinol Metab ; 316(5): E773-E781, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30860880

RESUMO

Preeclampsia (PE) is a major cause of maternal mortality and morbidity worldwide. Although there has been great progress in the understanding of PE, the exact cause for the disease development is still unclear. Recently, studies showed that genetic deletion of ELABELA (ELA, also known as APELA) could induce PE-like symptoms in mice. However, the role of ELA in the disease development of PE remains elusive. Our objective was to measure the changes of ELA levels in maternal serum, urine, and placenta from preeclamptic pregnant women and healthy pregnant women and evaluate the correlation between ELA levels and the occurrence of PE. Additionally, we investigated the effect of ELA on the migration and proliferation of human trophoblast cells. ELA levels are significantly decreased in late-onset PE pregnancies compared with normal pregnancies. The mRNA and protein expressions of ELA and the apelin receptor (APLNR or APJ) in late-onset PE placental tissues are also decreased. Furthermore, our in vitro study showed that the addition of ELA significantly increased the invasion ability and proliferation of trophoblast cells, which were inhibited by the APJ-specific antagonist ML221. Our study identified ELA as significantly decreased in late-onset PE; therefore, it might play an important role in the pathogenesis of late-onset PE.


Assuntos
Receptores de Apelina/genética , Hormônios Peptídicos/metabolismo , Placentação/fisiologia , Pré-Eclâmpsia/metabolismo , Adulto , Receptores de Apelina/metabolismo , Estudos de Casos e Controles , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Feminino , Humanos , Nitrobenzoatos/farmacologia , Hormônios Peptídicos/farmacologia , Placenta/metabolismo , Gravidez , Piranos/farmacologia , RNA Mensageiro/metabolismo , Trofoblastos/efeitos dos fármacos , Adulto Jovem
18.
EBioMedicine ; 41: 636-648, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30824385

RESUMO

BACKGROUND: Preeclampsia is a major complication of pregnancy with no medical treatment. It is associated with placental oxidative stress, hypoxia and inflammation leading to soluble fms-like tyrosine kinase 1 (sFlt-1) and soluble endoglin (sENG) secretion and reduced placental growth factor (PlGF). This results in widespread endothelial dysfunction causing hypertension and multisystem organ injury. Sulfasalazine is an anti-inflammatory and antioxidant medication used to treat autoimmune disease. Importantly, it is safe in pregnancy. We examined the potential of sulfasalazine to quench antiangiogenic factors and endothelial dysfunction and increase angiogenic factor secretion. METHODS: We performed functional experiments using primary human pregnancy tissues to examine the effects of sulfasalazine on sFlt-1, sENG and PlGF secretion. Sulfasalazine is known to inhibit nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) and upregulate heme-oxygenase 1 (HO-1) thus we explored the effect of these transcription factors on sFlt-1 secretion from human cytotrophoblasts. We examined the ability of sulfasalazine to reduce key markers of endothelial dysfunction and dilate whole blood vessels. FINDINGS: We demonstrate sulfasalazine administration reduces sFlt-1 and sENG and upregulates PlGF secretion from human placental tissues. Furthermore sulfasalazine mitigates endothelial dysfunction in several in vitro/ex vivo assays. It enhanced endothelial cell migration and proliferation, promoted blood vessel dilation (vessels obtained from women at caesarean section) and angiogenic sprouting from whole blood vessel rings. The effect of sulfasalazine on the secretion of sFlt-1 was not mediated through either the NFkB or HO-1 pathways. INTERPRETATION: We conclude that sulfasalazine reduces sFlt-1 and sENG secretion and endothelial dysfunction and upregulates PlGF. Sulfasalazine has potential to treat or prevent preeclampsia and warrants investigation in clinical trials. FUNDING: This work was funded by The National Health and Medical Research Council of Australia (NHMRC; #1048707, #1046484. #1101871, #1064845), an Arthur Wilson RANZCOG scholarship and a Norman Beischer Medical Research Foundation grant. FB was supported by a NHMRC Early Career Fellowship (NHMRC #1142636). NJH was supported by a CR Roper Research Fellowship. The NHMRC provided salary support (#1136418 to ST #1062418 to TKL, #1064845 to SS). The funders had no role in study design, data collection, analysis, decision to publish or the preparation of the manuscript.


Assuntos
Fator de Crescimento Placentário/metabolismo , Sulfassalazina/farmacologia , Regulação para Cima/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Endoglina/genética , Endoglina/metabolismo , Feminino , Heme Oxigenase-1/antagonistas & inibidores , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , NF-kappa B/metabolismo , Placenta/citologia , Fator de Crescimento Placentário/genética , Pré-Eclâmpsia/patologia , Gravidez , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Trofoblastos/citologia , Trofoblastos/efeitos dos fármacos , Trofoblastos/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo
19.
Environ Health Perspect ; 127(2): 27003, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30810372

RESUMO

BACKGROUND: Phthalates are environmental contaminants commonly used as plasticizers in polyvinyl chloride (PVC) products. Recently, exposure to phthalates has been associated with preterm birth, low birth weight, and pregnancy loss. There is limited information about the possible mechanisms linking maternal phthalate exposure and placental development, but one such mechanism may be mediated by peroxisome proliferator­activated receptor γ (PPARγ). PPARγ belongs to the nuclear receptor superfamily that regulates, in a ligand-dependent manner, the transcription of target genes. Studies of PPARγ-deficient mice have demonstrated its essential role in lipid metabolism and placental development. In the human placenta, PPARγ is expressed in the villous cytotrophoblast (VCT) and is activated during its differentiation into syncytiotrophoblast. OBJECTIVES: The goal of this study was to investigate the action of mono(2-ethylhexyl) phthalate (MEHP) on PPARγ activity during in vitro differentiation of VCTs. METHODS: We combined immunofluorescence, PPARγ activity/hCG assays, western blotting, and lipidomics analyses to characterize the impacts of physiologically relevant concentrations of MEHP (0.1, 1, and 10 µM) on cultured VCTs isolated from human term placentas. RESULTS: Doses of 0.1 and 1 µM MEHP showed significantly lower PPARγ activity and less VCT differentiation in comparison with controls, whereas, surprisingly, a 10 µM dose had the opposite effect. MEHP exposure inhibited hCG production and significantly altered lipid composition. In addition, MEHP had significant effects on the mitogen-activated protein kinase (MAPK) pathway. CONCLUSIONS: This study suggests that MEHP has a U-shaped dose­response effect on trophoblast differentiation that is mediated by the PPARγ pathway and acts as an endocrine disruptor in the human placenta. https://doi.org/10.1289/EHP3730.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Dietilexilftalato/análogos & derivados , Poluentes Ambientais/efeitos adversos , PPAR gama/genética , Trofoblastos/efeitos dos fármacos , Dietilexilftalato/efeitos adversos , Feminino , Humanos , Masculino , PPAR gama/metabolismo , Placenta/efeitos dos fármacos , Placenta/fisiologia , Gravidez , Trofoblastos/fisiologia
20.
Fertil Steril ; 111(3): 489-496.e5, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30709546

RESUMO

OBJECTIVE: To study the effects of insulin and metformin on primary trophoblasts from early pregnancies. DESIGN: Experimental in vitro study. SETTING: Academic research institute. PATIENT(S): Trophoblasts from healthy patients undergoing first trimester elective termination of pregnancy and primary lung fibroblasts (IMR-90). INTERVENTION(S): Culture and treatment with insulin and metformin of primary trophoblasts and primary lung fibroblasts (IMR-90). MAIN OUTCOME MEASURE(S): DNA damage measured by expression of γ-H2AX with immunofluorescence and Western blot. Apoptosis measured by expression of cleaved caspase-3 by Western blot. Cell survival measured by cell proliferation assay. RESULT(S): Culture of purified primary trophoblast cells in the presence of insulin at levels as low as 1 nM resulted in a 386% increase in the number of cell with elevated γ-H2AX expression, a 66% reduction in cell survival and a marked increase of cleaved caspase-3 expression. Pretreatment of trophoblasts with therapeutic doses of metformin prevented the detrimental effects of insulin. Treatment with insulin and/or metformin had no effects on primary fibroblasts. CONCLUSION(S): Elevated insulin levels are directly toxic to first trimester trophoblasts and result in increased DNA damage, apoptosis, and decreased cell survival. These effects are prevented by metformin. Trophoblast cells from early pregnancy are uniquely vulnerable to elevated levels of insulin. These findings, if confirmed in vivo, suggest that there may be a role for insulin resistance screening before attempting pregnancy and for focusing on prevention of hyperinsulinemia during early pregnancy.


Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Insulina/toxicidade , Metformina/farmacologia , Trofoblastos/efeitos dos fármacos , Biomarcadores/metabolismo , Caspase 3/metabolismo , Células Cultivadas , Citoproteção , Feminino , Histonas/metabolismo , Humanos , Gravidez , Primeiro Trimestre da Gravidez , Cultura Primária de Células , Trofoblastos/metabolismo , Trofoblastos/patologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA