RESUMEN
INTRODUCTION: Preeclampsia (PE) is associated with an exaggerated maternal systemic inflammatory response. Throughout gestation, the placenta releases extracellular vesicles through the syncytiotrophoblast layer (STB) into the maternal circulation and this is increased in PE. Expression of Siglec-6, a transmembrane receptor of molecular weight 50 KDa, is upregulated in PE placental tissue. METHODS: Here we investigated respective abundance of Siglec-6 in PE -and normal pregnancy- (NP) derived placental lysates (PL) and syncytiotrophoblast-derived extracellular vesicles (STBEV). STBEV from PE and NP placentas were isolated through dual-lobe placental perfusion and serial ultracentrifugation. Siglec-6 was characterized by immunohistochemistry, immunoblotting, mass spectrometry (MS), and deglycosylation. RESULTS: Immunoblotting revealed the expected Siglec-6 (50 KDa) band present in both PE and NP PL, however an additional heavier band was observed at 70 KDa only in PE PL, but not in NP. When interrogating STBEV we saw an absence of the expected 50 KDa band but the 70 KDa was present predominantly only in the PE STBEV. Deglycosylation of PL and STBEV from PE showed that the 70 KDa and the 50 KDa bands were reduced to 48 KDa, suggesting glycosylation. Both 48 KDa and 70 KDa bands were subjected to MS, confirming Siglec-6 expression in both. DISCUSSION: Our data shows that the inability to detect Siglec-6 in circulation might be due to the placenta secreting STBEV carrying a modified glycosylated form of Siglec-6 with a 70 KDa molecular weight, significantly and uniquely upregulated in PE STBEV.
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Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Vesículas Extracelulares/metabolismo , Lectinas/metabolismo , Placenta/metabolismo , Preeclampsia/metabolismo , Trofoblastos/metabolismo , Adulto , Femenino , Glicosilación , Humanos , Embarazo , Regulación hacia ArribaRESUMEN
Syncytiotrophoblast extracellular vesicles (STBEVs), released into the maternal circulation during pregnancy, have been shown to affect vascular function; however, the mechanism remains unknown. In rats, STBEVs were shown to reduce endothelium-mediated vasodilation via lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), a multi-ligand scavenger receptor that has been associated with vascular dysfunction. Recently, LOX-1 was shown to interact with the angiotensin II type 1 receptor (AT-1). We hypothesized that, in pregnant mice, STBEVs would impair vascular function via LOX-1 and would specifically affect angiotensin II responses. Uterine arteries from pregnant control (C57BL/6) and LOX-1 knockout (LOX-1KO) mice were isolated on gestational day (GD) 18.5. Endothelium-dependent (methylcholine (MCh); ± N(G)-Nitro-L-arginine methyl ester to assess nitric oxide (NO) contribution), and -independent (sodium nitroprusside) vasodilation, and vasoconstriction (angiotensin II; ± AT-1 [candesartan] or angiotensin II type 2 receptor (AT-2) [PD123.319] receptor antagonists; high potassium salt solution) responses were assessed using wire myography. AT-1 and AT-2 expression was analyzed using fluorescence microscopy. Human umbilical vein endothelial cells (HUVECs) were stimulated with STBEVs ± LOX-1 blocking antibody, and superoxide and peroxynitrite production were analyzed. Although MCh-induced vasodilation was decreased (P=0.0012), NO contribution to vasodilation was greater in LOX-1KO mice (P=0.0055). STBEVs delayed angiotensin II tachyphylaxis in arteries from control but not LOX-1KO mice (P<0.0001), while AT-1 and AT-2 expression was unchanged. STBEVs increased peroxynitrite production in HUVECs via LOX-1 (P=0.0091). In summary, LOX-1 deletion altered endothelium-mediated vasodilation, suggesting that LOX-1 contributes to vascular adaptations in pregnancy. STBEVs increased angiotensin II responsiveness and oxidative stress levels via LOX-1, suggesting that increased LOX-1 expression/activation or STBEVs could adversely affect vascular function and contribute to vascular complications of pregnancy.
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Células Endoteliales/metabolismo , Vesículas Extracelulares/metabolismo , Comunicación Paracrina , Receptores Depuradores de Clase E/metabolismo , Trofoblastos/metabolismo , Arteria Uterina/metabolismo , Vasoconstricción , Vasodilatación , Adulto , Animales , Células Endoteliales/efectos de los fármacos , Femenino , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Estrés Oxidativo , Ácido Peroxinitroso/metabolismo , Embarazo , Receptores de Angiotensina/metabolismo , Receptores Depuradores de Clase E/deficiencia , Receptores Depuradores de Clase E/genética , Transducción de Señal , Superóxidos/metabolismo , Arteria Uterina/citología , Arteria Uterina/efectos de los fármacos , Vasoconstricción/efectos de los fármacos , Vasoconstrictores/farmacología , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacologíaRESUMEN
OBJECTIVES: The liver X receptors (LXRs) and farnesoid X receptor (FXR) have been identified in human platelets. Ligands of these receptors have been shown to have nongenomic inhibitory effects on platelet activation by platelet agonists. This, however, seems contradictory with the platelet hyper-reactivity that is associated with several pathological conditions that are associated with increased circulating levels of molecules that are LXR and FXR ligands, such as hyperlipidemia, type 2 diabetes mellitus, and obesity. APPROACH AND RESULTS: We, therefore, investigated whether ligands for the LXR and FXR receptors were capable of priming platelets to the activated state without stimulation by platelet agonists. Treatment of platelets with ligands for LXR and FXR converted platelets to the procoagulant state, with increases in phosphatidylserine exposure, platelet swelling, reduced membrane integrity, depolarization of the mitochondrial membrane, and microparticle release observed. Additionally, platelets also displayed features associated with coated platelets such as P-selectin exposure, fibrinogen binding, fibrin generation that is supported by increased serine protease activity, and inhibition of integrin αIIbß3. LXR and FXR ligand-induced formation of coated platelets was found to be dependent on both reactive oxygen species and intracellular calcium mobilization, and for FXR ligands, this process was found to be dependent on cyclophilin D. CONCLUSIONS: We conclude that treatment with LXR and FXR ligands initiates coated platelet formation, which is thought to support coagulation but results in desensitization to platelet stimuli through inhibition of αIIbß3 consistent with their ability to inhibit platelet function and stable thrombus formation in vivo.
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Benzoatos/farmacología , Bencilaminas/farmacología , Coagulación Sanguínea/efectos de los fármacos , Plaquetas/efectos de los fármacos , Isoxazoles/farmacología , Receptores X del Hígado/agonistas , Activación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Receptores Citoplasmáticos y Nucleares/agonistas , Plaquetas/metabolismo , Señalización del Calcio/efectos de los fármacos , Micropartículas Derivadas de Células/efectos de los fármacos , Micropartículas Derivadas de Células/metabolismo , Ciclofilinas/sangre , Relación Dosis-Respuesta a Droga , Fibrina/metabolismo , Fibrinógeno/metabolismo , Humanos , Ligandos , Receptores X del Hígado/sangre , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Selectina-P/sangre , Fosfatidilserinas/sangre , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Especies Reactivas de Oxígeno/sangre , Receptores Citoplasmáticos y Nucleares/sangreRESUMEN
Excessive release of syncytiotrophoblast extracellular vesicles (STBMs) from the placenta into the maternal circulation may contribute to the systemic inflammation that is characteristic of pre-eclampsia (PE). Other intravascular cells types (platelets, leukocytes, red blood cells [RBCs], and endothelium) may also be activated and release extracellular vesicles (EVs). We developed a multicolor flow cytometry antibody panel to enumerate and phenotype STBMs in relation to other EVs in plasma from nonpregnant (NonP) and normal pregnant (NormP) women, and women with late-onset PE. Nanoparticle tracking analysis (NTA) was used to determine EV size and concentration. In vitro-derived STBMs and EVs from platelets, leukocytes, RBCs, and endothelial cells were examined to select suitable antibodies to analyze the corresponding plasma EVs. Flow cytometry analysis of plasma from NonP, NormP, and PE showed that STBMs comprised the smallest group of circulating EVs, whereas most were derived from platelets. The next most abundant group comprised unidentified orphan EVs (which did not label with any of the antibodies in the panel), followed by EVs from RBCs and leukocytes. NTA showed that the total number of EVs in plasma was significantly elevated in NormP and late-onset PE women compared to NonP controls, and that EVs were smaller in size. In general, EVs were elevated in pregnancy plasma apart from platelet EVs, which were reduced. These studies did not show any differences in EVs between NormP and PE, probably because late-onset PE was studied.
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Citometría de Flujo/métodos , Nanopartículas/análisis , Preeclampsia/sangre , Vesículas Secretoras , Trofoblastos , Adulto , Estudios de Casos y Controles , Rastreo Celular/métodos , Células Cultivadas , Color , Espacio Extracelular/química , Espacio Extracelular/metabolismo , Femenino , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Preeclampsia/metabolismo , Embarazo , Vesículas Secretoras/metabolismo , Trofoblastos/citología , Trofoblastos/metabolismo , Trofoblastos/ultraestructuraRESUMEN
Preeclampsia remains a significant obstetric risk worldwide. The pathophysiology of preeclampsia is complex, with multiple stages involving maladaptations in both placental and maternal physiology. The placenta links the pre-clinical stage of impaired remodeling of the uterine vasculature, occurring in early pregnancy, to the later clinical stages characterised by the maternal syndrome of hypertension and proteinuria. This review focuses on some of the recent candidates for the missing links in this process.
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Placenta/fisiopatología , Preeclampsia/fisiopatología , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Hipertensión/fisiopatología , Placenta/irrigación sanguínea , Embarazo , Proteinuria/fisiopatologíaRESUMEN
Cellular microvesicles and nanovesicles (exosomes) are involved in many disease processes and have major potential as biomarkers. However, developments in this area are constrained by limitations in the technology available for their measurement. Here we report on the use of fluorescence nanoparticle tracking analysis (NTA) to rapidly size and phenotype cellular vesicles. In this system vesicles are visualized by light scattering using a light microscope. A video is taken, and the NTA software tracks the brownian motion of individual vesicles and calculates their size and total concentration. Using human placental vesicles and plasma, we have demonstrated that NTA can measure cellular vesicles as small as ≈ 50 nm and is far more sensitive than conventional flow cytometry (lower limit ≈ 300 nm). By combining NTA with fluorescence measurement we have demonstrated that vesicles can be labeled with specific antibody-conjugated quantum dots, allowing their phenotype to be determined. FROM THE CLINICAL EDITOR: The authors of this study utilized fluorescence nanoparticle tracking analysis (NTA) to rapidly size and phenotype cellular vesicles, demonstrating that NTA is far more sensitive than conventional flow cytometry.
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Micropartículas Derivadas de Células/química , Micropartículas Derivadas de Células/ultraestructura , Nanopartículas/análisis , Nanotecnología/métodos , Placenta/citología , Micropartículas Derivadas de Células/genética , Femenino , Citometría de Flujo , Fluorescencia , Humanos , Tamaño de la Partícula , Fenotipo , EmbarazoRESUMEN
Syncytiotrophoblast derived Extracellular Vesicles (STBEV) from normal pregnancy (NP) have previously been shown to interact with circulating monocytes and B cells and induce pro-inflammatory cytokine release. Early-onset preeclampsia (EOPE) is associated with an exacerbated inflammatory response, yet there is little data regarding late-onset PE (LOPE) and immune function. Here, using a macrophage/monocyte cell line THP-1, we investigated the inflammatory potential of STBEV, comprising medium/large-STBEV (>200nm) and small-STBEV (<200nm), isolated from LOPE (n=6) and normal (NP) (n=6) placentae via dual-lobe ex-vivo placental perfusion and differential centrifugation. THP-1 cells bound and internalised STBEV isolated from NP and LOPE placentae, as revealed by flow cytometry, confocal microscopy, and ELISA. STBEV-treated THP-1 cells were examined for cytokine gene expression by RT-qPCR and the cell culture media examined for secreted cytokines/chemokines. As expected, NP medium/large-STBEV significantly upregulated the transcriptional expression of TNF-α, IL-10, IL-6, IL-12, IL-8 and TGF-ß compared to PE medium/large-STBEV. However, there was no significant difference in the small STBEV population between the two groups, although in general, NP small STBEVs slightly upregulated the same cytokines. In contrast, LOPE STBEV (medium and large) did not induce pro-inflammatory responses by differentiated THP-1 macrophages. This decreased effect of LOPE STBEV was echoed in cytokine/chemokine release. Our results appear to suggest that STBEV from LOPE placentae do not have a major immune-modulatory effect on macrophages. In contrast, NP STBEV caused THP-1 cells to release pro-inflammatory cytokines. Thus, syncytiotrophoblast extracellular vesicles from LOPE dampen immune functions of THP-1 macrophages, suggesting an alternative mechanism leading to the pro-inflammatory environment observed in LOPE.
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Vesículas Extracelulares/fisiología , Macrófagos/inmunología , Placenta/inmunología , Preeclampsia/inmunología , Trofoblastos/ultraestructura , Adulto , Citocinas/biosíntesis , Citocinas/genética , Femenino , Citometría de Flujo , Humanos , Microscopía Confocal , Microscopía Electrónica de Transmisión , Embarazo , Células THP-1RESUMEN
INTRODUCTION: The aim was to investigate syncytiotrophoblast extracellular vesicle (STBEV) uptake mechanisms by primary endothelial cells, the effects on gene expression, cell activation as well as the effect of aspirin. METHODS: The STBEVs were derived using the placental perfusion system, from normal or preeclamptic placentas. Endothelial uptake was analysed with flow cytometry. To elucidate uptake, different inhibitors were tested; Cytochalasin D, Chlorpromazine hydrochloride, Methyl-B-cyclodextrin, Dynasore and Wortmannin. Endothelial gene expression was evaluated using an endothelial cell biology qPCR array. Cell activation was studied by ICAM-1 surface expression after STBEV exposure, with and without aspirin treatment. RESULTS: Normal and preeclamptic STBEV uptake was blocked in similar ways. Chlorpromazine, Dynasore and Wortmannin almost completely blocked STBEV uptake. Methyl-B-cyclodextrin blocked 45-60% of the uptake while Cytochalasin D did not block uptake at all. Neither normal nor preeclamptic STBEVs had any significant effects on endothelial gene expression. Normal STBEVs down-regulated cell surface protein ICAM-1 expression, with and without aspirin treatment. Aspirin had no effect on STBEV uptake or cellular gene expression on its own, however it down regulated ICAM-1 protein expression in combination with preeclamptic STBEV exposure. DISCUSSION: STBEV uptake primarily occurred through clathrin-mediated endocytosis. The STBEVs had no significant effect on gene expression but did have effects on ICAM-1 surface expression. The prophylactic mechanisms of aspirin may be by preventing the endothelium from being activated by the preeclamptic STBEVs.
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Endocitosis , Células Endoteliales/fisiología , Vesículas Extracelulares/fisiología , Trofoblastos/fisiología , Adulto , Aspirina , Clatrina/metabolismo , Femenino , Expresión Génica , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Embarazo , Cultivo Primario de CélulasRESUMEN
Vascular complications in pregnancy (e.g. preeclampsia) are a major source of maternal and foetal morbidity and mortality, and may be due to excessive release of placental syncytiotrophoblast-derived extracellular vesicles (STBEVs) into the maternal circulation. Increased activity of the multi-ligand scavenger receptor Lectin-like Oxidized LDL Receptor-1 (LOX-1) is associated with vascular dysfunction, and LOX-1 has been shown to interact with angiotensin II receptor type 1 (AT1). We hypothesized that STBEVs contribute to vascular dysfunction via LOX-1 and AT1 receptors during pregnancy. Uterine arteries from late pregnant wildtype and LOX-1 overexpressing mice were incubated overnight with or without STBEVs and vascular function was assessed using wire myography. STBEV-incubation decreased angiotensin II responsiveness only in wildtype mice, which coincided with decreased AT1 contribution and expression. Thus, STBEVs reduced angiotensin II responsiveness in normal pregnancy, but not in conditions of increased LOX-1 expression, suggesting that STBEVs (via LOX-1) play a role in normal adaptations to pregnancy. Oxidized LDL (a LOX-1 ligand) increased angiotensin II-induced vasoconstriction in STBEV-incubated arteries from both mouse strains, suggesting that the LOX-1 pathway may be involved in complicated pregnancies with elevated STBEVs and oxidized LDL levels (such as preeclampsia). These data increase our understanding of vascular complications during pregnancy.
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Vesículas Extracelulares/metabolismo , Preeclampsia/metabolismo , Receptores Depuradores de Clase E/metabolismo , Trofoblastos/patología , Arteria Uterina/metabolismo , Animales , Células Cultivadas , Vesículas Extracelulares/patología , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Miografía , Circulación Placentaria , Embarazo , Receptor de Angiotensina Tipo 1/metabolismo , Receptores Depuradores de Clase E/genética , Arteria Uterina/patología , VasoconstricciónRESUMEN
Gestational diabetes mellitus (GDM) is the most common metabolic disorder in pregnancy and is characterized by insulin resistance and decreased circulating glucagon-like peptide-1 (GLP-1). GDM resolves rapidly after delivery implicating the placenta in the disease. This study examines the biological functions that cause this pathology. The placenta releases syncytiotrophoblast-derived extracellular vesicles (STB-EVs) into the maternal circulation, which is enhanced in GDM. Dipeptidyl peptidase IV (DPPIV) is known to play a role in type 2 diabetes by breaking down GLP-1, which in turn regulates glucose-dependent insulin secretion. STB-EVs from control and GDM women were analysed. We show that normal human placenta releases DPPIV-positive STB-EVs and that they are higher in uterine than paired peripheral blood, confirming placental origin. DPPIV-bound STB-EVs from normal perfused placentae are dose dependently inhibited with vildagliptin. DPPIV-bound STB-EVs from perfused placentae are able to breakdown GLP-1 in vitro. STB-EVs from GDM perfused placentae show greater DPPIV activity. Importantly, DPPIV-bound STB-EVs increase eightfold in the circulation of women with GDM. This is the first report of STB-EVs carrying a biologically active molecule that has the potential to regulate maternal insulin secretion.
RESUMEN
Fetal development may be compromised by adverse events at the placental interface between mother and fetus. However, it is still unclear how the communication between mother and fetus occurs through the placenta. In vitro - models of the human placental barrier, which could help our understanding and which recreate three-dimensional (3D) structures with biological functionalities and vasculatures, have not been reported yet. Here we present a 3D-vascularized human primary placental barrier model which can be constructed in 1 day. We illustrate the similarity of our model to first trimester human placenta, both in its structure and in its ability to respond to altered oxygen and to secrete factors that cause damage cells across the barrier including embryonic cortical neurons. We use this model to highlight the possibility that both the trophoblast and the endothelium within the placenta might play a role in the fetomaternal dialogue.
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Células del Tejido Conectivo/citología , Endotelio Vascular/citología , Placenta/irrigación sanguínea , Trofoblastos/citología , Células Cultivadas , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Neuronas/citología , Placenta/citología , EmbarazoRESUMEN
INTRODUCTION: Placental syncytiotrophoblast (STB) release extracellular vesicles (STB-EVs) that communicate physiological and pathological placental signals to the maternal organs. STB-EV release also increases in preeclampsia (PE). Here we explored the cargo of PP13 in STB-EVs from PE versus control placentas. METHODS: Placentae were harvested following cesarean section deliveries, and dual placental lobe perfusion was used to harvest STB-EV. Maternal side perfusate was centrifuged at 10,000â¯×â¯g to yield the STB microvesicles, and then at 150,000â¯×â¯g to yield STB exosomes. Total STB-EVs (tSTB-EVs) were collected using a one step 150,000â¯×â¯g centrifugation. Placental origin and size distribution were assessed by Western blotting and Nanoparticle Tracking Analysis, respectively. PP13 expression was determined by Western blot and ELISA. RESULTS: Placental alkaline phosphatase (PLAP; a STB specific marker) was present in all preparations. Total tSTB-EVs and STB-EXs also expressed the exosome markers such as the Apoptosis-Linked Gene 2-Interacting Protein X (Alix) and the cluster differentiation protein 9 (CD9). PP13 was localized to the outer surface and intra-vesicular compartments of all fractions. Surface to total PP13 ratios were â¼1:1 for all STB-EV preparations. In contrast to the previously reported higher circulating concentrations of soluble PP13 in PE, significantly lower levels of PP13, normalized to total vesicular protein, were observed in PE samples. PP13 reduction in all STB-EVs' sub-populations may be attributed to differences in gestational age (GA). A simple correction for GA suggested that PE may be an important influence. CONCLUSIONS: PP13 is located in and on all types of STB-EVs. Circulating PP13 may therefore be either soluble or associated with extracellular vesicles with different pathophysiological effects in the maternal circulation.
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Galectinas/metabolismo , Preeclampsia/metabolismo , Proteínas Gestacionales/metabolismo , Trofoblastos/metabolismo , Adulto , Biomarcadores/metabolismo , Estudios de Casos y Controles , Endocitosis , Exosomas/metabolismo , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/ultraestructura , Femenino , Edad Gestacional , Humanos , Modelos Biológicos , Tamaño de la Partícula , Placenta/metabolismo , Embarazo , Transporte de Proteínas , Trofoblastos/ultraestructuraRESUMEN
The release of extracellular vesicles (EV) by the syncytiotrophoblast (STB) may be an important mechanism by which the placenta signals to the mother. STB derived EV (STBEV) are comprised predominantly of exosomes (50-150nm) and microvesicles (100-1000nm) that contain bioactive mediators such as proteins, nucleic acids and lipids. They, along with larger syncytial nuclear aggregates are released by the STB into the maternal circulation throughout gestation in normal pregnancy where they appear to have an immunoregulatory role, inhibiting T cell and NK cell responses. In pre-eclampsia (PE) STBEV are released in significantly increased numbers and have pro-inflammatory, anti-angiogenic and procoagulant activity, implicating them in the maternal systemic inflammation, endothelial dysfunction and activation of the clotting system which typifies the disorder. Research has focused on understanding the biological significance of STBEV by measuring their size and repertoire of molecules carried and how they differ in normal pregnancy and PE, using techniques such as Nanoparticle Tracking Analysis, flow cytometry and mass spectrometry. We have also found alterations in STBEV surface glycans associated with PE. The goal is to better understand the role STBEV play in normal pregnancy and PE and whether they are potential biomarkers of placental pathology and therapeutic targets in PE.
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Vesículas Extracelulares/metabolismo , Mediadores de Inflamación/metabolismo , Inflamación/inmunología , Células Asesinas Naturales/inmunología , Preeclampsia/inmunología , Linfocitos T/inmunología , Trofoblastos/metabolismo , Coagulación Sanguínea , Femenino , Humanos , Inmunomodulación , Espectrometría de Masas , Nanopartículas , Circulación Placentaria , Preeclampsia/terapia , Embarazo , Trofoblastos/patologíaRESUMEN
The ability to directly monitor the status of the placenta throughout pregnancy would be a major advance in both general and personalized obstetric care, allowing treatments to be tailored to the dynamic changes that can occur in gestation. Syncytiotrophoblast extracellular vesicles (STBEV) are membrane bound vesicles, released from the surface of the placenta directly into the maternal circulation, in the form of exosomes, microvesicles and apoptotic bodies. They carry many syncytiotrophoblast derived factors such as proteins, lipids, glycans and nucleic acids, which together could dynamically signal to the mother the status of the placenta. We review STBEV research and discuss the potential for STBEV to be used as circulating syncytiotrophoblast biopsies, accessible via a simple blood sample throughout pregnancy, giving a real-time readout of syncytiotrophoblast health. We also highlight advances in the use of extracellular vesicles as circulating tumour derived biopsies in the field of cancer research, which could prove beneficial to obstetric care.
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Vesículas Extracelulares/metabolismo , Placenta/metabolismo , Trofoblastos/metabolismo , Biopsia , Femenino , Estado de Salud , Humanos , EmbarazoRESUMEN
Fluorescence nanoparticle tracking analysis (fl-NTA) allows for accurate sizing, counting, and phenotyping of extracellular vesicles (EV). Here, we present two protocols for the analysis of EVs using fl-NTA, highlighting the potential pitfalls and challenges. The first protocol utilizes CellMask Orange™ (CMO) as a general membrane marker to label EVs derived from plasma. The second protocol describes the use of a Qdot-conjugated antibody to identify syncytiotrophoblast (STB)-derived EVs. "Standard" preparations of STB-derived EVs enriched for either microvesicles (STBMV) or exosomes (STBEX), containing a known amount of EV positive for the STB specific antigen placental alkaline phosphatase (PLAP), were also used to optimize fl-NTA camera settings.
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Vesículas Extracelulares , Nanopartículas , Espectrometría de Fluorescencia/métodos , Anticuerpos , Micropartículas Derivadas de Células , Exosomas/química , Vesículas Extracelulares/química , Nanopartículas/química , Puntos Cuánticos , Trofoblastos/metabolismoRESUMEN
During the pregnancy associated syndrome preeclampsia (PE), there is increased release of placental syncytiotrophoblast extracellular vesicles (STBEVs) and free foetal haemoglobin (HbF) into the maternal circulation. In the present study we investigated the uptake of normal and PE STBEVs by primary human coronary artery endothelial cells (HCAEC) and the effects of free HbF on this uptake. Our results show internalization of STBEVs into primary HCAEC, and transfer of placenta specific miRNAs from STBEVs into the endoplasmic reticulum and mitochondria of these recipient cells. Further, the transferred miRNAs were functional, causing a down regulation of specific target genes, including the PE associated gene fms related tyrosine kinase 1 (FLT1). When co-treating normal STBEVs with HbF, the miRNA deposition is altered from the mitochondria to the ER and the cell membrane becomes ruffled, as was also seen with PE STBEVs. These findings suggest that STBEVs may cause endothelial damage and contribute to the endothelial dysfunction typical for PE. The miRNA mediated effects on gene expression may contribute to the oxidative and endoplasmic reticulum stress described in PE, as well as endothelial reprogramming that may underlay the increased risk of cardiovascular disease reported for women with PE later in life.
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Células Endoteliales/metabolismo , Vesículas Extracelulares/metabolismo , MicroARNs/metabolismo , Placenta/metabolismo , Trofoblastos/metabolismo , Transporte Biológico , Micropartículas Derivadas de Células/metabolismo , Micropartículas Derivadas de Células/ultraestructura , Células Cultivadas , Vesículas Extracelulares/ultraestructura , Femenino , Humanos , MicroARNs/genética , Microscopía Confocal , EmbarazoRESUMEN
Syncytiotrophoblast extracellular vesicles (STBEVs) are placenta derived particles that are released into the maternal circulation during pregnancy. Abnormal levels of STBEVs have been proposed to affect maternal vascular function. The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a multi-ligand scavenger receptor. Increased LOX-1 expression and activation has been proposed to contribute to endothelial dysfunction. As LOX-1 has various ligands, we hypothesized that, being essentially packages of lipoproteins, STBEVs are able to activate the LOX-1 receptor thereby impairing vascular function via the production of superoxide and decreased nitric oxide bioavailability. Uterine arteries were obtained in late gestation from Sprague-Dawley rats and incubated for 24h with or without human STBEVs (derived from a normal pregnant placenta) in the absence or presence of a LOX-1 blocking antibody. Vascular function was assessed using wire myography. Endothelium-dependent maximal vasodilation to methylcholine was impaired by STBEVs (MCh Emax: 57.7±5.9% in STBEV-incubated arteries vs. 77.8±2.9% in controls, p<0.05). This was prevented by co-incubation of STBEV-incubated arteries with LOX-1 blocking antibodies (MCh Emax: 78.8±4.3%, p<0.05). Pre-incubation of the vessels with a nitric oxide synthase inhibitor (L-NAME) demonstrated that the STBEV-induced impairment in vasodilation was due to decreased nitric oxide contribution (ΔAUC 12.2±11.7 in STBEV-arteries vs. 86.5±20 in controls, p<0.05), which was abolished by LOX-1 blocking antibody (ΔAUC 98.9±17, p<0.05). In STBEV-incubated vessels, LOX-1 inhibition resulted in an increased endothelial nitric oxide synthase expression (p<0.05), to a level similar to control vessels. The oxidant scavenger, superoxide dismutase, did not improve this impairment, nor were vascular superoxide levels altered. Our data support an important role for STBEVs in impairment of vascular function via activation of LOX-1 and reduced nitric oxide mediated vasodilation. Moreover, we postulate that the LOX-1 pathway could be a potential therapeutic target in pathologies associated with vascular dysfunction during pregnancy.
Asunto(s)
Vesículas Extracelulares/fisiología , Receptores Depuradores de Clase E/fisiología , Trofoblastos/citología , Arteria Uterina/fisiología , Vasodilatación , Animales , Femenino , Humanos , Embarazo , Ratas , Ratas Sprague-DawleyRESUMEN
Preeclampsia, a multisystem hypertensive disorder of pregnancy, is associated with increased systemic vascular resistance. Placentae from patients with preeclampsia have reduced levels of endothelial nitric oxide synthase (eNOS) and, thus, less nitric oxide (NO). Syncytiotrophoblast extracellular vesicles (STBEV), comprising microvesicles (STBMV) and exosomes, carry signals from the syncytiotrophoblast to the mother. We hypothesized that STBEV-bound eNOS (STBEV-eNOS), capable of producing NO, are released into the maternal circulation. Dual-lobe ex vivo placental perfusion and differential centrifugation was used to isolate STBEV from preeclampsia (n=8) and normal pregnancies (NP; n=11). Plasma samples of gestational age-matched preeclampsia and NP (n=6) were used to isolate circulating STBMV. STBEV expressed placental alkaline phosphatase, confirming placental origin. STBEV coexpressed eNOS, but not inducible nitric oxide synthase, confirmed using Western blot, flow cytometry, and immunodepletion. STBEV-eNOS produced NO, which was significantly inhibited by N G-nitro-l-arginine methyl ester (eNOS inhibitor; P<0.05) but not by N-(3-(aminomethyl) bezyl) acetamidine) (inducible nitric oxide synthase inhibitor). STBEV-eNOS catalytic activity was confirmed by visualizing eNOS dimerization. STBEV-eNOS was more abundant in uterine vein compared with peripheral blood, indicating placental origin. STBEV isolated from preeclampsia-perfused placentae had lower levels of STBEV-eNOS (STBMV; P<0.05) and overall lower NO activity (STBMV, not significant; syncytiotrophoblast extracellular exosomes, P<0.05) compared with those from NP. Circulating plasma STBMV from preeclampsia women had lower STBEV-eNOS expression compared with that from NP women (P<0.01). This is the first observation of functional eNOS expressed on STBEV from NP and preeclampsia placentae, as well as in plasma. The lower STBEV-eNOS NO production seen in preeclampsia may contribute to the decreased NO bioavailability in this disease.
Asunto(s)
Vesículas Extracelulares/fisiología , Hipertensión , Óxido Nítrico Sintasa de Tipo III/metabolismo , Preeclampsia , Trofoblastos , Adulto , Determinación de la Presión Sanguínea/métodos , Células Cultivadas , Femenino , Humanos , Hipertensión/diagnóstico , Hipertensión/etiología , Óxido Nítrico/metabolismo , Preeclampsia/metabolismo , Preeclampsia/patología , Preeclampsia/fisiopatología , Embarazo , Estadística como Asunto , Trofoblastos/patología , Trofoblastos/fisiología , Resistencia Vascular/fisiologíaRESUMEN
PROBLEM: We have previously found that C1q is constitutively expressed by invading trophoblast and endothelial cells of decidua and contributes to vascular and tissue remodeling. Based on these findings, we sought to determine whether there were changes in the circulating level of C1q that may be used as a diagnostic and predictive marker of preeclampsia. METHOD OF STUDY: We measured the levels of C1q, C4, and complement activation products in serum or plasma of normal pregnant women and preeclamptic patients from different cohorts. RESULTS: We observed a marked decrease in the concentration of C1q associated with a reduced level of C4 in preeclamptic patients as compared to matched healthy pregnant woman but no significant difference in the circulating level of the activating products C5a and the soluble terminal complement complex sC5b-9. Analysis of serum samples collected at early phase of pregnancy from women who later developed preeclampsia failed to show a decrease in C1q level. CONCLUSION: The results of the present investigation demonstrate that low levels of C1q and C4 are associated with preeclampsia but cannot be used as predictive markers.
Asunto(s)
Complemento C1q/metabolismo , Preeclampsia/sangre , Preeclampsia/diagnóstico , Adulto , Biomarcadores/sangre , Estudios de Casos y Controles , Activación de Complemento , Complemento C4/metabolismo , Complemento C5a/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Femenino , Edad Gestacional , Humanos , Preeclampsia/inmunología , Preeclampsia/patología , EmbarazoRESUMEN
PROBLEM: The pregnancy-associated disease preeclampsia is related to the release of syncytiotrophoblast extracellular vesicles (STBEV) by the placenta. To improve functional research on STBEV, reliable and specific methods are needed to quantify them. However, only a few quantification methods are available and accepted, though imperfect. For this purpose, we aimed to provide an enzyme-linked sorbent assay (ELSA) to quantify STBEV in fluid samples based on their microvesicle characteristics and placental origin. METHOD OF STUDY: Ex vivo placenta perfusion provided standards and samples for the STBEV quantification. STBEV were captured by binding of extracellular phosphatidylserine to immobilized annexin V. The membranous human placental alkaline phosphatase on the STBEV surface catalyzed a colorimetric detection reaction. RESULTS AND CONCLUSION: The described ELSA is a rapid and simple method to quantify STBEV in diverse liquid samples, such as blood or perfusion suspension. The reliability of the ELSA was proven by comparison with nanoparticle tracking analysis.