RESUMEN

Placental trophoblasts form the interface between the fetal and maternal environments and serve to limit the maternal-fetal spread of viruses. Here we show that cultured primary human placental trophoblasts are highly resistant to infection by a number of viruses and, importantly, confer this resistance to nonplacental recipient cells by exosome-mediated delivery of specific microRNAs (miRNAs). We show that miRNA members of the chromosome 19 miRNA cluster, which are almost exclusively expressed in the human placenta, are packaged within trophoblast-derived exosomes and attenuate viral replication in recipient cells by the induction of autophagy. Together, our findings identify an unprecedented paracrine and/or systemic function of placental trophoblasts that uses exosome-mediated transfer of a unique set of placental-specific effector miRNAs to directly communicate with placental or maternal target cells and regulate their immunity to viral infections.

Asunto(s)

Autofagia/genética , Cromosomas Humanos Par 19/genética , Resistencia a la Enfermedad/genética , MicroARNs/genética , Placenta/citología , Trofoblastos/virología , Virosis/transmisión , Análisis de Varianza , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Exosomas/genética , Exosomas/metabolismo , Femenino , Proteínas Fluorescentes Verdes , Humanos , Placenta/metabolismo , Embarazo , Trofoblastos/metabolismo

RESUMEN

Placental hypoperfusion causes cellular hypoxia and is associated with fetal growth restriction and preeclampsia. In response to hypoxia, the repertoire of genes expressed in placental trophoblasts changes, which influences key cellular processes such as differentiation and fusion. Diverse miRNAs were recently found to modulate the cellular response to hypoxia. Here we show that miR-424, which was previously shown to be upregulated by hypoxia in nontrophoblastic cell types, is uniquely downregulated in primary human trophoblasts by hypoxia or chemicals known to hinder cell differentiation. We also identify FGFR1 as a direct target of miR-424 in human trophoblasts. This effect is unique to miR-424 and is not seen with other members of this miRNA family that are expressed in trophoblasts, such as miR-15 and miR-16. Our findings establish a unique role for miR-424 during differentiation of human trophoblasts.

Asunto(s)

Hipoxia/metabolismo , MicroARNs/metabolismo , Placenta/metabolismo , Trofoblastos/metabolismo , Diferenciación Celular/genética , Línea Celular Tumoral , Células Cultivadas , Regulación hacia Abajo , Femenino , Humanos , MAP Quinasa Quinasa 1/genética , MAP Quinasa Quinasa 1/metabolismo , MicroARNs/genética , Placenta/citología , Embarazo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Trofoblastos/citología

RESUMEN

In this study we tested whether plasma from preeclamptic women contains factors that can activate endothelial cells in the presence of monocytes in vitro. Plasma from preeclamptic women (n=6), healthy pregnant women (n=6) and nonpregnant women (n=6) was incubated with mono-cultures and co-cultures of human umbilical vein endothelial cells (HUVEC) and monomac-6 monocytes. Reactive oxygen species (ROS) production and ICAM-1 expression were measured using flow cytometry. Whether scavenging of ROS by superoxide dismutase and catalase inhibited HUVEC ICAM-1 expression was also investigated. We found that in HUVEC co-cultured with monomac-6 cells but not in HUVEC cultured alone, ICAM-1 was upregulated after incubation with plasma from preeclamptic women but not plasma from non-pregnant women. Also in co-cultures, monomac-6 ICAM-1 was upregulated by plasma from preeclamptic women, while in both mono- and co-cultures monomac-6 ROS production was upregulated by plasma from pregnant and preeclamptic women, compared with plasma from non-pregnant women. Scavenging of ROS by superoxide dismutase and catalase resulted in a further upregulation of HUVEC ICAM-1 after incubation with plasma from preeclamptic women, compared with incubation without superoxide dismutase and catalase. These results show that endothelial cells in vitro are activated by plasma of preeclamptic women only if they are co-cultured with monocytes. This upregulation appeared not to be due to extracellular ROS production by monocytes or HUVEC, pointing to involvement of other mechanisms. Our data suggest that plasma of preeclamptic women activates monocytes, and that these monocytes subsequently activate endothelial cells.

Asunto(s)

Células Endoteliales/metabolismo , Monocitos/inmunología , Plasma/inmunología , Preeclampsia/sangre , Catalasa/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Femenino , Citometría de Flujo , Expresión Génica , Humanos , Molécula 1 de Adhesión Intercelular/biosíntesis , Molécula 1 de Adhesión Intercelular/genética , Monocitos/metabolismo , Reacción en Cadena de la Polimerasa , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/metabolismo , Cordón Umbilical/citología

RESUMEN

OBJECTIVE: Plasma hemopexin activity, associated with increased vascular permeability, was evaluated in healthy pregnant and non-pregnant women and in pre-eclamptic women. METHODS: Hemopexin activity and the hemopexin inhibitor, extracellular ATP, were assayed in plasma from pregnant (n = 10), preeclamptic (n = 9), and non-pregnant women (n = 10) using standard methods. Abdominal fascia tissue fragments from preeclamptic and pregnant women were immunohistochemically stained for vascular ecto-apyrase or ecto-5'nucleotidase. RESULTS: The data show significantly enhanced Hx activity exclusively in plasma from pregnant women and significantly enhanced plasma ATP in pre-eclamptic women compared with the other groups. Dephosphorylation of preeclamptic plasma resulted in reactivation of Hx activity. Fascia tissue-samples from preeclamptic women showed reduced ecto-apyrase activity and enhanced ecto-5'nucleotidase activity compared to pregnant women. CONCLUSION: Enhanced hemopexin activity may be associated with normal pregnancy, but not with preeclampsia. Decreased hemopexin in pre-eclamptic patients may be due to enhanced plasma ATP, which is possibly promoted by diminished activity of vascular ecto-apyrase.

Asunto(s)

Hemopexina/metabolismo , Preeclampsia/metabolismo , 5'-Nucleotidasa/metabolismo , Adenosina Trifosfato/metabolismo , Adulto , Animales , Antígenos CD/metabolismo , Apirasa/metabolismo , Biomarcadores/sangre , Biopsia , Permeabilidad Capilar , Estudios de Casos y Controles , Cesárea , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiopatología , Fascia/patología , Femenino , Mesangio Glomerular/metabolismo , Mesangio Glomerular/fisiopatología , Humanos , Inmunohistoquímica , Países Bajos , Circulación Placentaria , Preeclampsia/patología , Preeclampsia/fisiopatología , Embarazo , Ratas

RESUMEN

Endogenous microRNAs (miRNAs) post-transcriptionally regulate mRNA and protein expression during tissue development and function. Whereas adaptation to environmental insults are tightly regulated in human tissues, the role of miRNAs and miRNA biogenesis proteins in this context is inadequately explored. We sought to analyse the expression of the key RNAi enzyme Argonaute2 (Ago2) and other miRNA biogenesis proteins in human trophoblasts during differentiation and in hypoxic environment. Using an in vitro analysis of primary term human trophoblasts, we identified the expression of the core miRNA biogenesis proteins in human villous trophoblasts, with expression levels unaffected by cellular differentiation. We found that the miRNA biosynthetic pathway was functional and produced miRNAs, with miR-93 up-regulated and miR-424 down-regulated in hypoxic environment. In contrast, hypoxia did not alter the expression of key miRNA machinery proteins. The pivotal miRNA processing enzyme Ago2, along with its interacting protein DP103, were expressed in normal placentas as well as in placentas from pregnancies complicated by placental hypoperfusion that resulted in fetal growth restriction. Ago2 and DP103 co-immunoprecipitated, and did not limit trophoblast response to hypoxic stress. We concluded that the core miRNA machinery proteins are expressed and functional in human trophoblasts. The influence of hypoxia on the expression of a subset of placental miRNA species is unlikely to reflect altered expression of key miRNA biogenesis proteins.

Asunto(s)

Hipoxia de la Célula , MicroARNs/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Interferencia de ARN , Complejo Silenciador Inducido por ARN/metabolismo , Trofoblastos/metabolismo , Proteínas Argonautas , Hipoxia de la Célula/genética , Células Cultivadas , Proteína 20 DEAD-Box , ARN Helicasas DEAD-box/metabolismo , Factor 2 Eucariótico de Iniciación , Femenino , Perfilación de la Expresión Génica , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Factores de Iniciación de Péptidos/genética , ARN Mensajero/metabolismo , Complejo Silenciador Inducido por ARN/genética , Factores de Tiempo

RESUMEN

OBJECTIVE: At present it is unclear whether endothelial activation is systematically present in preeclampsia or restricted to specialized vascular beds. Therefore, this study aimed to investigate the presence of generalized proinflammatory endothelial activation in severe, early-onset preeclampsia in vivo. METHODS: During caesarean section, biopsies were obtained from abdominal subcutaneous fat, abdominal fascia, and myometrium from 11 severe, early-onset preeclamptic and 19 healthy pregnant women. Prior to caesarean, section plasma levels of von Willebrand Factor (vWF), sVCAM-1, and C-reactive protein (CRP) were measured by ELISA. Consecutive cryostat sections were stained immunohistochemically for CD31, E-selectin, VCAM-1, and ICAM-1. For subcutaneous fat tissue, endothelial gene expression levels of E-selectin, VCAM-1, ICAM-1, endothelin-1 (ET-1), and endothelial nitric oxide synthase (eNOS) were quantified by real-time RT-PCR, using normalization to the endothelium-specific housekeeping genes CD31 and VE-cadherin. RESULTS: Plasma levels of vWF, sVCAM-1, and CRP were elevated in the preeclampsia group compared to the control group, indicating enhanced endothelial activation and inflammatory response in the severely diseased preeclamptic women. By immunohistochemical analysis, no E-selectin and VCAM-1 expression could be detected in, and no differences in endothelial ICAM-1 staining could be observed between the preeclampsia and the control group for all tissues studied. Endothelial gene expression levels of E-selectin, VCAM-1, ICAM-1, ET-1, and eNOS were comparable between the preeclampsia and control group. CONCLUSION: Protein and gene expression analysis of E-selectin, VCAM-1, ICAM-1, ET-1, and eNOS, key mediators involved in pro-inflammatory endothelial activation, could not identify endothelial activation in severe, early-onset preeclampsia in the tissues studied. However, elevated plasma levels of markers of endothelial activation and inflammation were observed. These results may suggest that in severe, early-onset preeclampsia pro-inflammatory endothelial cell activation is not a generalized phenomenon, but is likely restricted to (possibly organ-specific) specialized vascular beds.

Asunto(s)

Endotelio Vascular/fisiología , Inflamación , Preeclampsia/fisiopatología , Adulto , Biopsia , Selectina E/biosíntesis , Endotelina-1/biosíntesis , Femenino , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Molécula 1 de Adhesión Intercelular/biosíntesis , Preeclampsia/inmunología , Embarazo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Índice de Severidad de la Enfermedad , Molécula 1 de Adhesión Celular Vascular/biosíntesis

RESUMEN

OBJECTIVE: Systemic endothelial dysfunction is a central feature in the pathophysiology of preeclampsia. Its cell biologic and molecular basis is poorly understood. One leading hypothesis argues that endothelial dysfunction is caused by (at present largely unknown) circulating factors released from the ischemic placenta. This study investigated the effects of plasma factors of severe, early-onset preeclamptic women versus healthy pregnant women on endothelial gene expression in vitro. METHODS: Plasma samples were taken from eight severe early-onset preeclamptic women and eight matched pregnant control women. Primary human umbilical vein endothelial cell (HUVEC) and human glomerular microvascular endothelial cell (hGMEC) cultures were incubated with 20% (vol/vol) plasma for 4, 12, and 24 hours. Identical amounts of RNA isolated from HUVEC from three preeclamptic and three control samples were pooled for each time point, and subsequently hybridized on human 60-mer oligonucleotide microarrays containing 17,000 genes. Gene expression levels of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), interleukin-8 (IL-8), and interleukin-6 (IL-6) in HUVEC and hGMEC were quantified using real-time reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Microarray analyses of individual genes identified no genes that were up- or down-regulated more than 2.7-fold, and analyses of gene ontologies showed no gene ontology significantly up- or down-regulated in HUVEC by preeclamptic plasma. IL-8 gene expression was modestly induced by preeclamptic plasma after 4, 12, and 24 hours of HUVEC and hGMEC incubation, as identified by real-time RT-PCR. The other genes analyzed did not show altered regulation by preeclamptic plasma factors. CONCLUSIONS: In vitro, plasma from preeclamptic patients does not substantially alter endothelial gene expression profile. Only modest induction of IL-8 gene expression was observed. These results indicate that mechanisms other than soluble plasma constituents are likely involved in systemic endothelial cell activation in preeclampsia.

Asunto(s)

Endotelio Vascular/fisiología , Molécula 1 de Adhesión Intercelular/sangre , Interleucina-6/sangre , Interleucina-8/sangre , Preeclampsia/sangre , Molécula 1 de Adhesión Celular Vascular/sangre , Adulto , Antígenos/metabolismo , Proteína C-Reactiva/metabolismo , Endotelio Vascular/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Perfilación de la Expresión Génica , Humanos , Recién Nacido , Molécula 1 de Adhesión Intercelular/biosíntesis , Molécula 1 de Adhesión Intercelular/genética , Interleucina-6/biosíntesis , Interleucina-6/genética , Interleucina-8/biosíntesis , Interleucina-8/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Preeclampsia/genética , Embarazo , Segundo Trimestre del Embarazo , ARN Mensajero/biosíntesis , ARN Mensajero/química , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Venas Umbilicales , Molécula 1 de Adhesión Celular Vascular/biosíntesis , Molécula 1 de Adhesión Celular Vascular/genética , Factor de von Willebrand/inmunología