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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 , Pré-Eclâmpsia/metabolismo , Receptores Depuradores Classe E/metabolismo , Trofoblastos/patologia , Artéria Uterina/metabolismo , Animais , Células Cultivadas , Vesículas Extracelulares/patologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miografia , Circulação Placentária , Gravidez , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores Depuradores Classe E/genética , Artéria Uterina/patologia , VasoconstriçãoRESUMO
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.
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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 do Tecido Conjuntivo/citologia , Endotélio Vascular/citologia , Placenta/irrigação sanguínea , Trofoblastos/citologia , Células Cultivadas , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Neurônios/citologia , Placenta/citologia , GravidezRESUMO
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 Endoteliais/metabolismo , Vesículas Extracelulares/metabolismo , Comunicação Parácrina , Receptores Depuradores Classe E/metabolismo , Trofoblastos/metabolismo , Artéria Uterina/metabolismo , Vasoconstrição , Vasodilatação , Adulto , Animais , Células Endoteliais/efeitos dos fármacos , Feminino , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estresse Oxidativo , Ácido Peroxinitroso/metabolismo , Gravidez , Receptores de Angiotensina/metabolismo , Receptores Depuradores Classe E/deficiência , Receptores Depuradores Classe E/genética , Transdução de Sinais , Superóxidos/metabolismo , Artéria Uterina/citologia , Artéria Uterina/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
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 , Pré-Eclâmpsia/metabolismo , Proteínas da Gravidez/metabolismo , Trofoblastos/metabolismo , Adulto , Biomarcadores/metabolismo , Estudos de Casos e Controles , Endocitose , Exossomos/metabolismo , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/ultraestrutura , Feminino , Idade Gestacional , Humanos , Modelos Biológicos , Tamanho da Partícula , Placenta/metabolismo , Gravidez , Transporte Proteico , Trofoblastos/ultraestruturaRESUMO
Disturbances in endoplasmic reticulum (ER) function lead to ER stress which, when severe or prolonged, may result in apoptosis. Severe ER stress has been implicated in several pathological conditions including pre-eclampsia, a multisystem disorder of pregnancy associated with the release of pro-inflammatory factors from the placenta into the maternal circulation. Here, we show that severe ER stress induced by two distinct mechanisms in BeWo choriocarcinoma cells leads to the release of extracellular vesicles (EVs) carrying pro-inflammatory damage-associated molecular pattern (DAMP) molecules. Co-treatment with the antioxidant pyrrolidine dithiocarbamate results in a reduction in ER stress-induced EV-associated DAMP release. We further demonstrate that severe ER stress is associated with changes in the expression of several stress-related proteins, notably Cited-2 and phosphorylated JNK. Together, these data indicate that severe ER stress-mediated release of EV-associated DAMPs may contribute to the heightened systemic maternal inflammatory response characteristic of pre-eclampsia and may also be relevant to other chronic inflammatory diseases which display elevated ER stress.
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PROBLEM: Procalcitonin (PCT) is the prohormone of calcitonin which is usually released from neuroendocrine cells of the thyroid gland (parafollicular) and the lungs (K cells). PCT is synthesized by almost all cell types and tissues, including monocytes and parenchymal tissue, upon LPS stimulation. To date, there is no evidence for PCT expression in the placenta both in physiological and pathological conditions. METHOD: Circulating and placental PCT levels were analysed in pre-eclamptic (PE) and control patients. Placental cells and macrophages (PBDM), stimulated with PE sera, were analysed for PCT expression. The effect of anti-TNF-α antibody was analysed. RESULTS: Higher PCT levels were detected in PE sera and in PE placentae compared to healthy women. PE trophoblasts showed increased PCT expression compared to those isolated from healthy placentae. PE sera induced an upregulation of PCT production in macrophages and placental cells. The treatment of PBDM with PE sera in the presence of anti-TNF-α completely abrogated the effect induced by pathologic sera. CONCLUSION: Trophoblast cells are the main producer of PCT in PE placentae. TNF-α, in association with other circulating factors present in PE sera, upregulates PCT production in macrophages and normal placental cells, thus contributing to the observed increased in circulating PCT in PE sera.
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Calcitonina/metabolismo , Macrófagos/imunologia , Placenta/metabolismo , Pré-Eclâmpsia/metabolismo , Gravidez , Trofoblastos/metabolismo , Adulto , Estudos de Coortes , Feminino , Humanos , Placenta/patologia , Trofoblastos/patologia , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima , Adulto JovemRESUMO
Brain injury elicits a systemic acute-phase response (APR), which is responsible for co-ordinating the peripheral immunological response to injury. To date, the mechanisms responsible for signalling the presence of injury or disease to selectively activate responses in distant organs were unclear. Circulating endogenous extracellular vesicles (EVs) are increased after brain injury and have the potential to carry targeted injury signals around the body. Here, we examined the potential of EVs, isolated from rats after focal inflammatory brain lesions using IL-1ß, to activate a systemic APR in recipient naïve rats, as well as the behavioural consequences of EV transfer. Focal brain lesions increased EV release, and, following isolation and transfer, the EVs were sequestered by the liver where they initiated an APR. Transfer of blood-borne EVs from brain-injured animals was also enough to suppress exploratory behaviours in recipient naïve animals. EVs derived from brain endothelial cell cultures treated with IL-1ß also activated an APR and altered behaviour in recipient animals. These experiments reveal that inflammation-induced circulating EVs derived from endothelial cells are able to initiate the APR to brain injury and are sufficient to generate the associated sickness behaviours, and are the first demonstration that EVs are capable of modifying behavioural responses.
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Reação de Fase Aguda/metabolismo , Encefalite/metabolismo , Encefalite/fisiopatologia , Células Endoteliais/metabolismo , Vesículas Extracelulares/metabolismo , Comportamento de Doença , Animais , Comportamento Animal , Citocinas/metabolismo , Modelos Animais de Doenças , Encefalite/etiologia , Encefalite/patologia , Hepatite/etiologia , Hepatite/metabolismo , Hepatite/patologia , Mediadores da Inflamação/metabolismo , Células de Kupffer/metabolismo , Masculino , RatosRESUMO
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 Endoteliais/metabolismo , Vesículas Extracelulares/metabolismo , MicroRNAs/metabolismo , Placenta/metabolismo , Trofoblastos/metabolismo , Transporte Biológico , Micropartículas Derivadas de Células/metabolismo , Micropartículas Derivadas de Células/ultraestrutura , Células Cultivadas , Vesículas Extracelulares/ultraestrutura , Feminino , Humanos , MicroRNAs/genética , Microscopia Confocal , GravidezRESUMO
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.
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Vesículas Extracelulares/fisiologia , Receptores Depuradores Classe E/fisiologia , Trofoblastos/citologia , Artéria Uterina/fisiologia , Vasodilatação , Animais , Feminino , Humanos , Gravidez , Ratos , Ratos Sprague-DawleyRESUMO
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.
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Vesículas Extracelulares/fisiologia , Hipertensão , Óxido Nítrico Sintase Tipo III/metabolismo , Pré-Eclâmpsia , Trofoblastos , Adulto , Determinação da Pressão Arterial/métodos , Células Cultivadas , Feminino , Humanos , Hipertensão/diagnóstico , Hipertensão/etiologia , Óxido Nítrico/metabolismo , Pré-Eclâmpsia/metabolismo , Pré-Eclâmpsia/patologia , Pré-Eclâmpsia/fisiopatologia , Gravidez , Estatística como Assunto , Trofoblastos/patologia , Trofoblastos/fisiologia , Resistência Vascular/fisiologiaRESUMO
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 , Biópsia , Feminino , Nível de Saúde , Humanos , GravidezRESUMO
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 da Inflamação/metabolismo , Inflamação/imunologia , Células Matadoras Naturais/imunologia , Pré-Eclâmpsia/imunologia , Linfócitos T/imunologia , Trofoblastos/metabolismo , Coagulação Sanguínea , Feminino , Humanos , Imunomodulação , Espectrometria de Massas , Nanopartículas , Circulação Placentária , Pré-Eclâmpsia/terapia , Gravidez , Trofoblastos/patologiaRESUMO
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.
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Complemento C1q/metabolismo , Pré-Eclâmpsia/sangue , Pré-Eclâmpsia/diagnóstico , Adulto , Biomarcadores/sangue , Estudos de Casos e Controles , Ativação do Complemento , Complemento C4/metabolismo , Complemento C5a/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Feminino , Idade Gestacional , Humanos , Pré-Eclâmpsia/imunologia , Pré-Eclâmpsia/patologia , GravidezRESUMO
Pre-eclampsia (PE) complicates around 3% of all pregnancies and is one of the most common causes of maternal mortality worldwide. The pathophysiology of PE remains unclear however its underlying cause originates from the placenta and manifests as raised blood pressure, proteinuria, vascular or systemic inflammation and hypercoagulation in the mother. Women who develop PE are also at significantly higher risk of subsequently developing cardiovascular (CV) disease. In PE, the failing endoplasmic reticulum, oxidative and inflammatory stressed syncytiotrophoblast layer of the placenta sheds increased numbers of syncytiotrophoblast extracellular vesicles (STBEV) into the maternal circulation. Platelet reactivity, size and concentration are also known to be altered in some women who develop PE, although the underlying reasons for this have not been determined. In this study we show that STBEV from disease free placenta isolated ex vivo by dual placental perfusion associate rapidly with platelets. We provide evidence that STBEV isolated from normal placentas cause platelet activation and that this is increased with STBEV from PE pregnancies. Furthermore, treatment of platelets with aspirin, currently prescribed for women at high risk of PE to reduce platelet aggregation, also inhibits STBEV-induced reversible aggregation of washed platelets. Increased platelet reactivity as a result of exposure to PE placenta derived STBEVs correlates with increased thrombotic risk associated with PE. These observations establish a possible direct link between the clotting disturbances of PE and dysfunction of the placenta, as well as the known increased risk of thromboembolism associated with this condition.
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Plaquetas/fisiologia , Placenta/fisiopatologia , Pré-Eclâmpsia/sangue , Pré-Eclâmpsia/fisiopatologia , Trofoblastos/fisiologia , Adulto , Aspirina/farmacologia , Plaquetas/patologia , Estudos de Casos e Controles , Vesículas Extracelulares/patologia , Vesículas Extracelulares/fisiologia , Feminino , Humanos , Microscopia Eletrônica de Transmissão , Placenta/patologia , Ativação Plaquetária , Agregação Plaquetária/efeitos dos fármacos , Pré-Eclâmpsia/patologia , Gravidez , Trombose/etiologia , Trofoblastos/patologiaRESUMO
BACKGROUND: The innate immune system contributes to the outcome after stroke, where neuroinflammation and post-stroke systemic immune depression are central features. Tumor necrosis factor (TNF), which exists in both a transmembrane (tm) and soluble (sol) form, is known to sustain complex inflammatory responses associated with stroke. We tested the effect of systemically blocking only solTNF versus blocking both tmTNF and solTNF on infarct volume, functional outcome and inflammation in focal cerebral ischemia. METHODS: We used XPro1595 (a dominant-negative inhibitor of solTNF) and etanercept (which blocks both solTNF and tmTNF) to test the effect of systemic administration on infarct volume, functional recovery and inflammation after focal cerebral ischemia in mice. Functional recovery was evaluated after one, three and five days, and infarct volumes at six hours, 24 hours and five days after ischemia. Brain inflammation, liver acute phase response (APR), spleen and blood leukocyte profiles, along with plasma microvesicle analysis, were evaluated. RESULTS: We found that both XPro1595 and etanercept significantly improved functional outcomes, altered microglial responses, and modified APR, spleen T cell and microvesicle numbers, but without affecting infarct volumes. CONCLUSIONS: Our data suggest that XPro1595 and etanercept improve functional outcome after focal cerebral ischemia by altering the peripheral immune response, changing blood and spleen cell populations and decreasing granulocyte infiltration into the brain. Blocking solTNF, using XPro1595, was just as efficient as blocking both solTNF and tmTNF using etanercept. Our findings may have implications for future treatments with anti-TNF drugs in TNF-dependent diseases.
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Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/metabolismo , Recuperação de Função Fisiológica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismo , Animais , Injeções Intravenosas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Distribuição Aleatória , Resultado do Tratamento , Fator de Necrose Tumoral alfa/administração & dosagemRESUMO
INTRODUCTION: Optical techniques are routinely used to size and count extracellular vesicles (EV). For comparison of data from different methods and laboratories, suitable calibrators are essential. A suitable calibrator must have a refractive index (RI) as close to that of EV as possible but the RI of EV is currently unknown. To measure EV, RI requires accurate knowledge of size and light scattering. These are difficult to measure as most EVs cannot be resolved by light microscopy and their diameter is smaller than the wavelength of visible light. However, nanoparticle tracking analysis (NTA) provides both size and relative light scattering intensity (rLSI) values. We therefore sought to determine whether it was possible to use NTA to measure the RI of individual EVs. METHODS: NTA was used to measure the rLSI and size of polystyrene and silica microspheres of known size and RI (1.470 and 1.633, respectively) and of EV isolated from a wide range of cells. We developed software, based on Mie scattering code, to calculate particle RI from the rLSI data. This modelled theoretical scattering intensities for polystyrene and silica microspheres of known size (100 and 200 nm) and RI. The model was verified using data from the polystyrene and silica microspheres. Size and rLSI data for each vesicle were processed by the software to generate RI values. RESULTS: The following modal RI measurements were obtained: fresh urinary EV 1.374, lyophilised urinary EV 1.367, neuroblastoma EV 1.393, blood EV 1.398, EV from activated platelets 1.390, small placental EV 1.364-1.375 and 1.398-1.414 for large placental EV (>200 nm). Large placental EV had a significantly higher RI than small placental EV (p<0.0001). The spread of RI values was narrower for small EV than for the more heterogeneous large EV. DISCUSSION: Using NTA and Mie scattering theory, we have demonstrated that it is possible to estimate the RI of sub-micron EV using NTA data. EV typically had a modal RI of 1.37-1.39, whereas values of >1.40 were observed for some large (>200 nm) microvesicles. CONCLUSION: This method for measuring EV RI will be useful for developing appropriate calibrators for EV measurement.
RESUMO
Some psychiatric diseases in children and young adults are thought to originate from adverse exposures during foetal life, including hypoxia and hypoxia/reoxygenation. The mechanism is not understood. Several authors have emphasised that the placenta is likely to play an important role as the key interface between mother and foetus. Here we have explored whether a first trimester human placenta or model barrier of primary human cytotrophoblasts might secrete factors, in response to hypoxia or hypoxia/reoxygenation, that could damage neurones. We find that the secretions in conditioned media caused an increase of [Ca(2+)]i and mitochondrial free radicals and a decrease of dendritic lengths, branching complexity, spine density and synaptic activity in dissociated neurones from embryonic rat cerebral cortex. There was altered staining of glutamate and GABA receptors. We identify glutamate as an active factor within the conditioned media and demonstrate a specific release of glutamate from the placenta/cytotrophoblast barriers invitro after hypoxia or hypoxia/reoxygenation. Injection of conditioned media into developing brains of P4 rats reduced the numerical density of parvalbumin-containing neurones in cortex, hippocampus and reticular nucleus, reduced immunostaining of glutamate receptors and altered cellular turnover. These results show that the placenta is able to release factors, in response to altered oxygen, that can damage developing neurones under experimental conditions.
Assuntos
Encéfalo , Meios de Cultivo Condicionados/efeitos adversos , Hipóxia , Neurônios/efeitos dos fármacos , Oxigênio/farmacologia , Placenta/química , Animais , Animais Recém-Nascidos , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/patologia , Hipóxia Celular/fisiologia , Células Cultivadas , Córtex Cerebral/citologia , Meios de Cultivo Condicionados/química , Dendritos/efeitos dos fármacos , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Feminino , Feto , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Hipóxia/tratamento farmacológico , Hipóxia/patologia , Hipóxia/fisiopatologia , Potenciais da Membrana/efeitos dos fármacos , Neurônios/citologia , Neurônios/fisiologia , Placenta/citologia , Gravidez , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Técnicas de Cultura de TecidosRESUMO
BACKGROUND: Cell-free foetal haemoglobin (HbF) has been shown to play a role in the pathology of preeclampsia (PE). In the present study, we aimed to further characterize the harmful effects of extracellular free haemoglobin (Hb) on the placenta. In particular, we investigated whether cell-free Hb affects the release of placental syncytiotrophoblast vesicles (STBMs) and their micro-RNA content. METHODS: The dual ex-vivo perfusion system was used to perfuse isolated cotyledons from human placenta, with medium alone (control) or supplemented with cell-free Hb. Perfusion medium from the maternal side of the placenta was collected at the end of all perfusion phases. The STBMs were isolated using ultra-centrifugation, at 10,000×g and 150,000×g (referred to as 10K and 150K STBMs). The STBMs were characterized using the nanoparticle tracking analysis, identification of surface markers and transmission electron microscopy. RNA was extracted and nine different micro-RNAs, related to hypoxia, PE and Hb synthesis, were selected for analysis by quantitative PCR. RESULTS: All micro-RNAs investigated were present in the STBMs. Mir-517a, mir-141 and mir-517b were down regulated after Hb perfusion in the 10K STBMs. Furthermore, Hb was shown to be carried by the STBMs. CONCLUSION: This study showed that Hb perfusion can alter the micro-RNA content of released STBMs. Of particular interest is the alteration of two placenta specific micro-RNAs; mir-517a and mir-517b. We have also seen that STBMs may function as carriers of Hb into the maternal circulation.
Assuntos
Hemoglobinas/metabolismo , MicroRNAs/genética , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/metabolismo , Vesículas Transportadoras/metabolismo , Trofoblastos/metabolismo , Transporte Biológico , Espaço Extracelular/metabolismo , Feminino , Humanos , Gravidez , Vesículas Transportadoras/ultraestrutura , Trofoblastos/ultraestruturaRESUMO
OBJECTIVES: Cell injury releases actin, the most abundant cell protein. Gelsolin and vitamin D binding protein (VDBP) together depolymerise and clear cell-free actin. Impaired actin clearance is associated with several diseases and correlates with clinical outcome. The actin scavenging system was investigated in pre-eclampsia (PE), a procoagulant and proinflammatory state with placental and vascular damage. STUDY DESIGN: Plasma gelsolin and actin free VDBP (AFVDBP) were measured in PE (early onset <33weeks; late onset ≥36weeks), matched normal pregnant (normP) and non-pregnant (nonPr) women, using commercially available ELISAs. Longitudinal samples from normP and women who subsequently developed PE were also analysed. RESULTS: Plasma gelsolin fell during pregnancy (p=0.0002), with a concomitant rise in actin-free VDBP (p<0.001). Gelsolin concentrations were only significantly lower in established PE (p<0.05) when compared to non-pregnant controls. CONCLUSIONS: We have shown that the components of the actin clearance system, gelsolin and AFVDBP, are altered in normal pregnancy and further changes occur in established PE, suggesting depleted actin clearance in PE. Whether this is a cause or consequence of PE pathophysiology requires further investigation.