RESUMEN
The mechanisms of deficient placentation in the first trimester remain poorly understood, although apoptosis, hypoxia, and oxidative stress have been implicated. High uterine artery Doppler resistance indexes (RIs) are predictive of placental complications of pregnancy, such as preeclampsia, fetal growth restriction, and stillbirth. We provide evidence that even in the first trimester, pregnancies with high uterine artery Doppler RI demonstrate alterations in placental gene and protein expression. Apoptosis was significantly higher in high RI placental tissue, as determined by Western blot analysis of cleaved poly (ADP-ribose) polymerase and caspase 3. Protein expression of the trophoblast survival factor insulin-like growth factor-2 was significantly lower. Both high and normal RI placentas showed evidence of hypoxia and oxidative stress with expression of hypoxia-inducible factors 1α and 2α, heat shock protein 70, presence of nitrotyrosine residues, and lipid peroxidation. We observed no exaggerated placental hypoxia or oxidative stress associated with high RI pregnancies. High RI placental tissue demonstrated an altered balance of antioxidant enzyme activity. Hypoxia and oxidative stress appear to be a physiological state in early pregnancy; our data did not support the hypothesis that they are associated with deficient placentation in the first trimester. Higher levels of apoptosis, reduced insulin-like growth factor-2 expression, and altered antioxidant defenses may contribute to abnormal placentation and the later development of pregnancy complications, such as preeclampsia, fetal growth restriction, and stillbirth.
Asunto(s)
Apoptosis/fisiología , Oxígeno/metabolismo , Primer Trimestre del Embarazo/metabolismo , Trofoblastos/citología , Arteria Uterina/metabolismo , Útero/irrigación sanguínea , Adulto , Femenino , Humanos , Placenta/irrigación sanguínea , Placenta/metabolismo , Placentación/fisiología , Preeclampsia/metabolismo , Embarazo , Complicaciones del Embarazo/prevención & control , Adulto JovenRESUMEN
STUDY QUESTION: What are the effects of the eotaxin group of chemokines (CCL11, CCL24 and CCL26) on extravillous trophoblast (EVT) functions important during uterine decidual vessel remodelling? SUMMARY ANSWER: CCL11, CCL24 and CCL26 can regulate EVT migration, invasion and adhesion, highlighting a potential regulatory role for these chemokines during uterine decidual spiral arteriole remodelling in the first trimester of human pregnancy. WHAT IS KNOWN ALREADY: A successful human pregnancy depends on adequate remodelling of the uterine decidual spiral arterioles, a process carried out by EVT which invade from the placenta. The invasion by EVT into the maternal uterine decidual vessels is regulated by the interaction of many factors including members of the chemokine subfamily of cytokines. STUDY DESIGN, SIZE, DURATION: This study used the HTR8/SVneo cell line as a model for invasive EVT. All experiments were repeated on at least three separate occasions. PARTICIPANTS/MATERIALS, SETTING, METHODS: The effect of recombinant human CCL11, CCL24 and CCL26 on EVT migration and invasive potential was measured using the xCELLigence real-time system, wound-healing and Matrigel invasion assays, zymography to measure MMP activity and reverse zymography to measure TIMP activity. A commercially available adhesion assay was used to assess EVT adhesion to extracellular matrix proteins. MAIN RESULTS AND THE ROLE OF CHANCE: All the three eotaxins were found to significantly stimulate migration of the EVT-derived cell line HTR8/SVneo (P < 0.05) with no significant changes in cell number following treatment with each chemokine (P > 0.05). All the three eotaxins significantly increased HTR8/SVneo invasion (P < 0.05) and MMP2 activity (P < 0.05) without any effects on TIMP2 activity (P > 0.05). All the three eotaxins significantly increased HTR8/SVneo cell binding to collagen IV (P < 0.05) and fibronectin (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: This work has been conducted in vitro with a commonly used cell line model of EVT, HTR8/SVneo. WIDER IMPLICATIONS OF THE FINDINGS: This study is the first to comprehensively examine the effects of the eotaxin group of chemokines on EVT functions and demonstrates that all the three eotaxins have the ability to regulate EVT functions critical to their role in vessel remodelling. This identifies a new role for the eotaxin group of chemokines during placentation.
Asunto(s)
Quimiocina CCL11/farmacología , Quimiocina CCL24/farmacología , Quimiocinas CC/farmacología , Decidua/efectos de los fármacos , Trofoblastos/efectos de los fármacos , Arteriolas/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Línea Celular , Movimiento Celular/efectos de los fármacos , Quimiocina CCL11/fisiología , Quimiocina CCL24/fisiología , Quimiocina CCL26 , Quimiocinas CC/fisiología , Colágeno , Decidua/irrigación sanguínea , Combinación de Medicamentos , Femenino , Humanos , Laminina , Proteoglicanos , Trofoblastos/citologíaRESUMEN
During human pregnancy, natural killer (NK) cells accumulate in the maternal decidua, but their specific roles remain to be determined. Decidual NK (dNK) cells are present during trophoblast invasion and uterine spiral artery remodelling. These events are crucial for successful placentation and the provision of an adequate blood supply to the developing fetus. Remodelling of spiral arteries is impaired in the dangerous pregnancy complication pre-eclampsia. We studied dNK cells isolated from pregnancies at 9-14 weeks' gestation, screened by uterine artery Doppler ultrasound to determine resistance indices which relate to the extent of spiral artery remodelling. dNK cells were able to promote the invasive behaviour of fetal trophoblast cells, partly through HGF. Cells isolated from pregnancies with higher resistance indices were less able to do this and secreted fewer pro-invasive factors. dNK cells from pregnancies with normal resistance indices could induce apoptotic changes in vascular smooth muscle and endothelial cells in vitro, events of importance in vessel remodelling, partly through Fas signalling. dNK cells isolated from high resistance index pregnancies failed to induce vascular apoptosis and secreted fewer pro-apoptotic factors. We have modelled the cellular interactions at the maternal-fetal interface and provide the first demonstration of a functional role for dNK cells in influencing vascular cells. A potential mechanism contributing to impaired vessel remodelling in pregnancies with a higher uterine artery resistance is presented. These findings may be informative in determining the cellular interactions contributing to the pathology of pregnancy disorders where remodelling is impaired, such as pre-eclampsia.
Asunto(s)
Diferenciación Celular , Decidua/citología , Decidua/fisiología , Células Asesinas Naturales/fisiología , Embarazo/fisiología , Arteria Uterina/fisiología , Resistencia Vascular/fisiología , Apoptosis , Antígeno CD56/fisiología , Línea Celular , Movimiento Celular/fisiología , Células Cultivadas , Proteína Ligando Fas/fisiología , Femenino , Humanos , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Preeclampsia/patología , Preeclampsia/fisiopatología , Primer Trimestre del Embarazo/fisiología , Flujo Sanguíneo Regional/fisiología , Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Trofoblastos/citología , Trofoblastos/fisiología , Ultrasonografía , Arteria Uterina/diagnóstico por imagenRESUMEN
Remodeling of the uterine spiral arteries during pregnancy transforms them from high to low resistance vessels that lack vasoconstrictive properties. This process is essential to meet the demand for increased blood flow imposed by the growing fetus. Loss of endothelial and smooth muscle cells (SMC) is evident in remodeled arteries but the mechanisms underlying this transformation remain unknown. This study investigated the hypothesis that fetal trophoblast invading from the placenta instigate remodeling by triggering cell death in vascular SMC. Specifically, a role for trophoblast-derived death inducing cytokine tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) was investigated. Expression of the activating TRAIL receptors R1 and R2 was detected by flow cytometry on human aortic SMC and by immunohistochemistry on spiral artery SMC. Recombinant human TRAIL induced human aortic SMC apoptosis, which was inhibited by antibodies against TRAIL-R1 or -R2. Perfusion of denuded spiral artery segments with recombinant human TRAIL also induced SMC apoptosis. Trophoblasts isolated from first trimester placenta expressed membrane-associated TRAIL and induced apoptosis of human aortic SMC; apoptosis was significantly inhibited by a recombinant human TRAIL-R1:Fc construct. Trophoblast within the first trimester placental bed also expressed TRAIL. These data show that: 1) TRAIL causes SMC death; 2) trophoblast produce the apoptotic cytokine TRAIL; and 3) trophoblast induce SMC apoptosis via a TRAIL-dependent mechanism. We conclude that TRAIL produced by trophoblast causes apoptosis of SMC and thus may contribute to SMC loss during spiral artery remodeling in pregnancy.
Asunto(s)
Apoptosis/fisiología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Trofoblastos/metabolismo , Apoptosis/efectos de los fármacos , Arterias/citología , Células Cultivadas , Decidua/irrigación sanguínea , Decidua/citología , Femenino , Feto , Humanos , Microscopía por Video , Músculo Liso Vascular/citología , Miometrio/irrigación sanguínea , Miometrio/citología , Embarazo , Primer Trimestre del Embarazo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/biosíntesis , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Factores de Tiempo , Trofoblastos/citologíaRESUMEN
The pathologies of the pregnancy complications pre-eclampsia (PE) and fetal growth restriction (FGR) are established in the first trimester of human pregnancy. In a normal pregnancy, decidual spiral arteries are transformed into wide diameter, non-vasoactive vessels capable of meeting the increased demands of the developing fetus for nutrients and oxygen. Disruption of this transformation is associated with PE and FGR. Very little is known of how these first trimester changes are regulated normally and even less is known about how they are compromised in complicated pregnancies. Interactions between maternal and placental cells are essential for pregnancy to progress and this review will summarise the challenges in investigating this area. We will discuss how first trimester studies of pregnancies with an increased risk of developing PE/FGR have started to provide valuable information about pregnancy at this most dynamic and crucial time. We will discuss where there is scope to progress these studies further by refining the ability to identify compromised pregnancies at an early stage, by integrating information from many cell types from the same pregnancy, and by improving our methods for modelling the maternal-fetal interface in vitro.
Asunto(s)
Retardo del Crecimiento Fetal/etiología , Placentación , Preeclampsia/etiología , Primer Trimestre del Embarazo/fisiología , Investigación Biomédica/tendencias , Femenino , Humanos , EmbarazoRESUMEN
BACKGROUND: Chronic hypoxia-induced pulmonary hypertension is associated with increased pulmonary expression of nitric oxide synthase (NOS) enzymes. Nevertheless, some reports have indicated decreased pulmonary production of NO in the disease. To address this paradox, we determined pulmonary concentrations of the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA) in the hypoxia-induced pulmonary hypertension rat model. In addition, we determined whether dysregulation of the ADMA-metabolizing enzyme dimethylarginine dimethylaminohydrolase I (DDAH I) plays a role in this disease. METHODS AND RESULTS: Adult male rats were exposed for 1 week to either normoxia or hypoxia (10% oxygen). Lung tissues were used for Western blot analysis of endothelial NOS and DDAH I expression, measurement of lung NO and ADMA content, and in vitro assay of DDAH enzyme activity. Western blot analysis revealed a 1.9-fold increase in endothelial NOS protein and a 37% decrease in DDAH I protein in the lungs of hypoxia-exposed rats. Both pulmonary DDAH enzyme activity and NO content were significantly decreased in the hypoxic group (by 37% and 22%, respectively), but pulmonary ADMA concentrations were increased by 2.3-fold compared with the normoxic group. CONCLUSIONS: These data demonstrate that the rat chronic hypoxia-induced pulmonary hypertension model is associated with increased pulmonary concentrations of the NOS inhibitor ADMA. Moreover, pulmonary hypertensive rats exhibit reduced pulmonary expression and activity of the ADMA-metabolizing enzyme DDAH I. The decreased DDAH I and increased ADMA concentrations may therefore contribute to pulmonary hypertension via the competitive inhibition of pulmonary NOS enzymes.
Asunto(s)
Amidohidrolasas/metabolismo , Arginina/análogos & derivados , Hipertensión Pulmonar/fisiopatología , Hipoxia/fisiopatología , Animales , Arginina/metabolismo , Western Blotting , Enfermedad Crónica , Modelos Animales de Enfermedad , Activación Enzimática , Hipertensión Pulmonar/enzimología , Hipertensión Pulmonar/etiología , Hipoxia/complicaciones , Hipoxia/enzimología , Isoenzimas/metabolismo , Pulmón/irrigación sanguínea , Pulmón/química , Pulmón/metabolismo , Masculino , Nitratos/análisis , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo III , Nitritos/análisis , Ratas , Ratas Sprague-DawleyRESUMEN
OBJECTIVE: We investigated the hypothesis that antiphospholipid antibodies (aPL) have a detrimental effect on human extravillous trophoblast (EVT) differentiation into giant multinucleated cells "in vitro." DESIGN: The EVT were isolated from the placental chorion using enzymatic digestion and Percoll gradient centrifugation. After 24, 36, and 48 hours in culture, giant multinuclear cells (GMC) were identified by immunohistochemistry using antibodies to cytokeratin 7 and counted. SETTING: An academic research laboratory. PATIENT(S): Placentas were donated by women having an elective cesarean section for a normal pregnancy at term. MAIN OUTCOME MEASURE(S): This model was then used to investigate the effect of two different monoclonal aPL to beta2-glycoprotein 1 (IIC5 and ID2), and control mouse IgG antibody on EVT differentiation. RESULT(S): Freshly isolated EVT were nonproliferative but moved together losing their intervening cell walls and differentiated into GMC. Maximal numbers of GMC were detected after 48 hours of culture. The aPL, IIC5, and ID2 significantly inhibited GMC formation, whereas the mouse IgG control had no effect. CONCLUSION(S): Antiphospholipid antibodies can inhibit EVT differentiation and GMC formation "in vitro" suggesting that a failure of trophoblast differentiation and subsequent uteroplacental development may be an underlying pathology in antiphospholipid syndrome-associated pregnancy loss.
Asunto(s)
Anticuerpos Antifosfolípidos/farmacología , Trofoblastos/citología , Trofoblastos/inmunología , Anticuerpos Monoclonales/farmacología , Diferenciación Celular/inmunología , Células Cultivadas , Femenino , Células Gigantes/citología , Células Gigantes/inmunología , Humanos , Embarazo , Complicaciones del Embarazo/inmunología , Complicaciones del Embarazo/patologíaRESUMEN
Apoptosis induced by the death-inducing ligand FasL (CD95L) is a major mechanism of cell death. Trophoblast cells express the Fas receptor yet survive in an environment that is rich in the ligand. We report that basal nitric oxide (NO) production is responsible for the resistance of trophoblasts to FasL-induced apoptosis. In this study we demonstrate that basal NO production resulted in the inhibition of receptor clustering following ligand binding. In addition NO also protected cells through the selective nitrosylation, and inhibition, of protein kinase Cepsilon (PKCepsilon) but not PKCalpha. In the absence of NO production PKCepsilon interacted with, and phosphorylated, the anti-apoptotic protein cFLIP. The interaction is predominantly with the short form of cFLIP and its phosphorylation reduces its recruitment to the death-inducing signaling complex (DISC) that is formed following binding of a death-inducing ligand to its receptor. Inhibition of cFLIP recruitment to the DISC leads to increased activation of caspase 8 and subsequently to apoptosis. Inhibition of PKCepsilon using siRNA significantly reversed the sensitivity to apoptosis induced by inhibition of NO synthesis suggesting that NO-mediated inhibition of PKCepsilon plays an important role in the regulation of Fas-induced apoptosis.
Asunto(s)
Apoptosis , Proteína Ligando Fas/metabolismo , Óxido Nítrico/metabolismo , Compuestos Nitrosos/metabolismo , Proteína Quinasa C-epsilon/metabolismo , Receptor fas/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Inmunoprecipitación , Modelos Biológicos , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa/antagonistas & inhibidores , Fosforilación/efectos de los fármacos , Proteína Quinasa C-alfa/metabolismo , Proteína Quinasa C-epsilon/antagonistas & inhibidores , Interferencia de ARN , Trofoblastos/citología , Trofoblastos/efectos de los fármacos , Trofoblastos/enzimologíaRESUMEN
Hepatocyte growth factor (HGF) increases human trophoblast motility and invasion, an effect which is abrogated when inducible nitric oxide synthase (iNOS) is inhibited. In this study we have investigated the pathways involved in the regulation of trophoblast motility. Both basal and HGF-stimulated motility of the extravillous trophoblast cell line, SGHPL-4, were inhibited in a dose-dependent manner by the phosphatidylinositol-3-kinase (PI3-kinase) inhibitor, LY294002. HGF-stimulated iNOS expression was also inhibited by LY294002 and direct activation of PI3-kinase, using the peptide 740Y-P, led to an increase in iNOS expression and cell motility. Pretreatment with rapamycin, which acts at a point distal to PI3-kinase activation, also inhibited basal and HGF-stimulated motility. Inhibition of the p42/p44 mitogen activated protein kinase (MAPK) pathway but not the p38 MAPK pathway had significant inhibitory effects on HGF-stimulated but not basal trophoblast motility. Inhibition of p42/p44 MAPK also inhibited HGF-induced iNOS expression. This data demonstrate that the PI3-kinase signaling pathway is involved in basal trophoblast motility and that both MAPK and PI3-kinase signaling pathways are important in HGF-stimulated motility and iNOS expression.