Disruption of the Mouse Blood-Brain Barrier by Small Extracellular Vesicles from Hypoxic Human Placentas.

Cerebrovascular complications, including cerebral edema and ischemic and hemorrhagic stroke, constitute the leading cause of maternal mortality associated with preeclampsia. The underlying mechanisms of these cerebrovascular complications remain unclear. However, they are linked to placental dysfunction and blood-brain barrier (BBB) disruption. Nevertheless, the connection between these two distant organs is still being determined. Increasing evidence suggests that the placenta releases signaling molecules, including extracellular vesicles, into maternal circulation. Extracellular vesicles are categorized according to their size, with small extracellular vesicles (sEVs smaller than 200 nm in diameter) considered critical signaling particles in both physiological and pathological conditions. In preeclampsia, there is an increased number of circulating sEVs in maternal circulation, the signaling function of which is not well understood. Placental sEVs released in preeclampsia or from normal pregnancy placentas exposed to hypoxia induce brain endothelial dysfunction and disruption of the BBB. In this protocol, we assess whether sEVs isolated from placental explants cultured under hypoxic conditions (modeling one aspect of preeclampsia) disrupt the BBB in vivo.

Reduced Brain Cortex Angiogenesis in the Offspring of the Preeclampsia-Like Syndrome.

BACKGROUND: Children from pregnancies affected by preeclampsia have an increased risk of cognitive and behavioral alterations via unknown pathophysiology. We tested the hypothesis that preeclampsia generated reduced brain cortex angiogenesis in the offspring. METHODS: The preeclampsia-like syndrome (PELS) mouse model was generated by administering the nitric oxide inhibitor NG-nitroarginine methyl ester hydrochloride. Confirmatory experiments were done using 2 additional PELS models. While in vitro analysis used mice and human brain endothelial cells exposed to serum of postnatal day 5 pups or umbilical plasma from preeclamptic pregnancies, respectively. RESULTS: We report significant reduction in the area occupied by blood vessels in the motor and somatosensory brain cortex of offspring (postnatal day 5) from PELS compared with uncomplicated control offspring. These data were confirmed using 2 additional PELS models. Furthermore, circulating levels of critical proangiogenic factors, VEGF (vascular endothelial growth factor), and PlGF (placental growth factor) were lower in postnatal day 5 PELS. Also we found lower VEGF receptor 2 (KDR [kinase insert domain-containing receptor]) levels in mice and human endothelial cells exposed to the serum of postnatal day 5 PELS or fetal plasma of preeclamptic pregnancies, respectively. These changes were associated with lower in vitro angiogenic capacity, diminished cell migration, larger F-actin filaments, lower number of filopodia, and lower protein levels of F-actin polymerization regulators in brain endothelial cells exposed to serum or fetal plasma of offspring from preeclampsia. CONCLUSIONS: Offspring from preeclampsia exhibited diminished brain cortex angiogenesis, associated with lower circulating VEGF/PlGF/KDR protein levels, impaired brain endothelial migration, and dysfunctional assembly of F-actin filaments. These alterations may predispose to structural and functional alterations in long-term brain development.

Language Impairment in Children of Mothers with Gestational Diabetes, Preeclampsia, and Preterm Delivery: Current Hypothesis and Potential Underlying Mechanisms : Language Impartment and Pregnancy Complications.

Many conditions may impair or delay language development, including socioeconomic status, parent's education, or intrauterine environment. Accordingly, increasing evidence has described that pregnancy complications, including gestational diabetes mellitus (GDM), preeclampsia, and preterm delivery, are associated with the offspring's impaired neurodevelopment. Since language is one of the high brain functions, alterations in this function are another sign of neurodevelopment impairment. How these maternal conditions may generate language impairment has yet to be entirely understood. However, since language development requires adequate structural formation and function/connectivity of the brain, these processes must be affected by alterations in maternal conditions. However, the underlying mechanisms of these structural alterations are largely unknown. This manuscript critically analyzes the literature focused on the risk of developing language impairment in children of mothers with GDM, preeclampsia, and preterm delivery. Furthermore, we highlight potential underlying molecular mechanisms associated with these alterations, such as neuroinflammatory and metabolic and cerebrovascular alterations.

Brain Vascular Dysfunction in Mothers and Their Children Exposed to Preeclampsia.

Preeclampsia is a maternal syndrome characterized by the new onset of hypertension and proteinuria after 20 weeks of gestation associated with multisystemic complications, including brain alterations. Indeed, brain complications associated with preeclampsia are the leading direct causes of fetal and maternal morbidity and mortality, especially in low- and middle-income countries. In addition to the well-recognized long-term adverse cardiovascular effects of preeclampsia, women who have had preeclampsia have higher risk of stroke, dementia, intracerebral white matter lesions, epilepsy, and perhaps also cognitive decline postpartum. Furthermore, increasing evidence has also associated preeclampsia with similar cognitive and cerebral disorders in the offspring. However, the mechanistic links between these associations remain unresolved. This article summarizes the current knowledge about the cerebrovascular complications elicited by preeclampsia and the potential pathophysiological mechanisms involved, emphasizing the impaired brain vascular function in the mother and their offspring.

A robust quantitative approach for laser speckle contrast imaging perfusion analysis revealed anomalies in the brain blood flow in offspring mice of preeclampsia.

Microcirculation analysis of the brain cortex is challenging because surface perfusion varies rapidly in small space-time regions and is bone protected. The laser speckle contrast imaging (LSCI) technique allows analyzing in vivo brain vascular perfusion generating a large amount of data that requires sophisticated data analytics, making researchers invest much effort in processing. Our research question was whether the reduced placental perfusion model (RUPP) of preeclampsia (PE) was associated with impaired blood perfusion in the offspring's brains. We aimed to develop a robust numerical approach that mainly consisted of applying a signal-processing tool for calculating optimal segmentation and piece-wise fits of the offspring's brain perfusion signals obtained from the LSCI technique. We combined this tool with the usual statistical analysis, implementing both in Matlab software. We performed brain perfusion measurements from offspring (five days postnatal, P5) of control pregnant dams (sham, n = 13) and of RUPP dams (RUPP, n = 7) using the Pericam® PSI-HR system at a basal condition and after thermal stimuli (warm and cold). We found that pups of RUPP mice exhibited significant differences in perfusion and vascular response to thermal stimuli compared to the sham mice. These differences were associated with high data variability in the Sham group, while in the RUPP group, perfusion looks "stiffer." Data also suggest sex-dimorphism in the vascular response since female pups in the Sham group but not male pups showed statistically significant differences in response to the warm stimulus. Again, this sex-related difference was absent in pups of RUPP mice. In conclusion, we present a robust quantitative approach for LSCI measurements that revealed anomalies in the brain blood flow in offspring of the RUPP model of PE.

Dysregulation of vascular endothelial growth factor receptor 2 phosphorylation is associated with disruption of the blood-brain barrier and brain endothelial cell apoptosis induced by plasma from women with preeclampsia.

Disruption of the blood-brain barrier (BBB) is central in the pathophysiology of acute cerebral complications in women who have preeclampsia. Underling mechanisms are unclear. Using female human brain endothelial cells as an in vitro model of BBB, we show that plasma of women with preeclampsia increases cell apoptosis and permeability via activation of the vascular endothelial growth factor receptor 2 (VEGFR2). Since plasma of women with preeclampsia also enhanced VEGFR2 phosphorylation in the tyrosine 951 but decreased phosphorylation at the tyrosine 1175, we propose the former would be the more likely active form of VEGFR2 responsible for BBB alterations.

Cerebral Biomarkers and Blood-Brain Barrier Integrity in Preeclampsia.

Cerebral complications in preeclampsia contribute substantially to maternal mortality and morbidity. There is a lack of reliable and accessible predictors for preeclampsia-related cerebral complications. In this study, plasma from women with preeclampsia (n = 28), women with normal pregnancies (n = 28) and non-pregnant women (n = 16) was analyzed for concentrations of the cerebral biomarkers neurofilament light (NfL), tau, neuron-specific enolase (NSE) and S100B. Then, an in vitro blood−brain barrier (BBB) model, based on the human cerebral microvascular endothelial cell line (hCMEC/D3), was employed to assess the effect of plasma from the three study groups. Transendothelial electrical resistance (TEER) was used as an estimation of BBB integrity. NfL and tau are proteins expressed in axons, NSE in neurons and S100B in glial cells and are used as biomarkers for neurological injury in other diseases such as dementia, traumatic brain injury and hypoxic brain injury. Plasma concentrations of NfL, tau, NSE and S100B were all higher in women with preeclampsia compared with women with normal pregnancies (8.85 vs. 5.25 ng/L, p < 0.001; 2.90 vs. 2.40 ng/L, p < 0.05; 3.50 vs. 2.37 µg/L, p < 0.001 and 0.08 vs. 0.05 µg/L, p < 0.01, respectively). Plasma concentrations of NfL were also higher in women with preeclampsia compared with non-pregnant women (p < 0.001). Higher plasma concentrations of the cerebral biomarker NfL were associated with decreased TEER (p = 0.002) in an in vitro model of the BBB, a finding which indicates that NfL could be a promising biomarker for BBB alterations in preeclampsia.

Disruption of the Blood-Brain Barrier by Extracellular Vesicles From Preeclampsia Plasma and Hypoxic Placentae: Attenuation by Magnesium Sulfate.

[Figure: see text].

Alteraciones en el discurso narrativo en hijos de madres con preeclampsia / Alterations in the narrative discourse in children born to preeclamptic mothers

Antecedentes y objetivo: Evidencias epidemiológicas han mostrado que la descendencia de mujeres que tuvieron preeclampsia (hipertensión del embarazo) tienen mayor riesgo a desarrollar enfermedades cardiovasculares y alteraciones neurocognitivas, incluyendo en esta última categoría alteraciones del lenguaje. Sin embargo, el análisis de las alteraciones del lenguaje en niños nacidos de embarazos con preeclampsia es limitado, por lo tanto, el presente estudio tiene como objetivo investigar el lenguaje oral en hijos de madres con preeclampsia. Método: Se realiza un estudio transversal, previamente aprobado por el Comité Ético Científico del Hospital Clínico Herminda Martin de Chillán, Chile. A partir de un universo objetivo de 56 niños, se concretó el análisis en 17 menores, los cuales, a su vez, fueron subdivididos en: cuatro niños nacidos de embarazos normales y tres nacidos de embarazos preeclámpticos en edad preescolar; cinco niños nacidos de embarazos normales y cinco de embarazos preeclámpticos en edad escolar. Se evaluaron de manera general (cribado) los distintos niveles del lenguaje y el discurso narrativo utilizando una adaptación de los test disponibles en la literatura. Resultados: El puntaje total en la evaluación del lenguaje no mostró diferencias estadísticamente significativas en los niños de edad preescolar o escolar de ambos grupos de embarazos. Sin embargo, en la evaluación más detallada del discurso narrativo, tanto en su vertiente expresiva como comprensiva, se detectaron diferencias puntuales que se traducen en que un menor porcentaje de niños hijos de madres con preeclampsia en etapa preescolar no logran comprender o expresar parte del mensaje entregado (final de un cuento) en relación con sus respectivos controles por edad. Conclusiones: Los resultados indican que los niños hijos de madres con preeclampsia podrían tener una disminución de la comprensión y expresión del discurso narrativo en la edad preescolar.(AU)

Background and objective: Much epidemiological evidence has shown that the offspring born to preeclamptic women (hypertension in pregnancy) have a high risk for developing cardiovascular diseases and neurocognitive disorders, including language disorders later in life. Nevertheless, the analysis of language disorders in children born to preeclamptic women is limited. Therefore, the present study aims to investigate oral language in children of mothers who had preeclampsia. Method: This is a cross-sectional study, previously approved by the Scientific Ethics Committee of the Hospital Clínico Herminda Martin of Chillán, Chile. The analysis was performed on a third (n = 17) of a target universe of 56 children. These were sub-divided into groups of pre-school children born to normal pregnancies (n = 4) and preeclamptic women (n = 3); and children of school age born to normal (n = 5) and preeclamptic women (n = 5). We evaluated in general (screening) the different levels of language and narrative discourse using an adaptation of the tests available in the literature. Results: No statistically significant differences between children of pre-school or school age from both groups of pregnancies were found in the total score of the language evaluation test. However, in the more detailed analysis of either the expressive or the comprehensive aspect, specific differences were detected that resulted in a low percentage of children born to mothers with pre-eclampsia in the pre-school stage who failed to understand or express part of the delivered message (end of a tale) compared to the respective controls. Conclusions: The results indicate that children born to mothers with preeclampsia may have decreased understanding and expression of narrative discourse at preschool age. We suggest that preeclampsia might cause diminished oral verbal ability in offspring.(AU)

Preeclampsia and Increased Permeability Over the Blood-Brain Barrier: A Role of Vascular Endothelial Growth Receptor 2.

BACKGROUND: Cerebral complications in preeclampsia are leading causes of maternal mortality worldwide but pathophysiology is largely unknown and a challenge to study. Using an in vitro model of the human blood-brain barrier (BBB), we explored the role of vascular endothelial growth factor receptor 2 (VEGFR2) in preeclampsia. METHODS: The human brain endothelial cell line (hCMEC/D3) cultured on Tranwells insert was exposed (12 hours) to plasma from women with preeclampsia (n = 28), normal pregnancy (n = 28), and nonpregnant (n = 16) controls. Transendothelial electrical resistance (TEER) and permeability to 70 kDa fluorescein isothiocyanate (FITC)-dextran were measured for the assessment of BBB integrity. We explored possible underlying mechanisms, with a focus on the expression of tight junction proteins and phosphorylation of 2 tyrosine residues of VEGFR2, associated with vascular permeability and migration (pY951) and cell proliferation (pY1175). Plasma concentrations of soluble FMS-like tyrosine kinase-1 (sFlt-1) and placental growth factor (PlGF) were also measured. RESULTS: hCMEC/D3 exposed to plasma from women with preeclampsia exhibited reduced TEER and increased permeability to 70 kDa FITC-dextran. These cells upregulated the messenger ribonucleic acid (mRNA) levels of VEGFR2, and pY951-VEGFR2, but reduced pY1175-VEGFR2 (P < 0.05 in all cases). No difference in mRNA expression of tight junction protein was observed between groups. There was no correlation between angiogenic biomarkers and BBB permeability. CONCLUSIONS: We present a promising in vitro model of the BBB in preeclampsia. Selective tyrosine phosphorylation of VEGFR2 may participate in the increased BBB permeability in preeclampsia irrespective of plasma concentrations of angiogenic biomarkers.

Vascular endothelial growth factor and poor prognosis after ischaemic stroke.

BACKGROUND AND PURPOSE: Systemic inflammation conveys information about ischaemic stroke prognosis. Growth factors with neurotrophic and angiogenesis-regulating properties might provide additional information about sequelae. The prognostic performance of circulating vascular endothelial growth factor (VEGF), placental growth factor, interleukin 6 and C-reactive protein measured after acute ischaemic stroke was evaluated. METHODS: Blood samples were collected from n = 45 patients within 24-48 h of acute ischaemic stroke. The primary outcome was death or moderate to severe disability at 6 months (modified Rankin Scale >2). Logistic regression models were used to determine the area under the receiver operating characteristic curve (AUC). Correlation and principal component analyses were performed to examine interrelationships amongst biomarkers. RESULTS: Vascular endothelial growth factor was elevated in ischaemic stroke patients who died or had moderate to severe disability at six months. Correlation analysis revealed interrelationships between VEGF and HbA1c, triglycerides, erythrocyte sedimentation rate and National Institutes of Health Stroke Scale and Rankin scores, whereas principal component analyses identified VEGF as a major loading factor that discriminated good from poor prognosis. There were no significant differences in AUC using each protein individually to identify patients who had modified Rankin Scale score >2 at 6 months (n = 15/41, AUC 0.61-0.74). However, the AUC increased significantly when combining VEGF with interleukin 6 and C-reactive protein compared to the VEGF-only model (AUC 0.92 vs. 0.67, p = 0.02). CONCLUSION: Circulating VEGF was elevated 24-48 h after acute ischaemic stroke and conveyed prognostic information about moderate to severe disability at 6 months.

Advantages in Wound Healing Process in Female Mice Require Upregulation A2A-Mediated Angiogenesis under the Stimulation of 17ß-Estradiol.

Estrogenic steroids and adenosine A2A receptors promote the wound healing and angiogenesis processes. However, so far, it is unclear whether estrogen may regulate the expression and pro-angiogenic activity of A2A receptors. Using in vivo analyses, we showed that female wild type (WT) mice have a more rapid wound healing process than female or male A2A-deficient mice (A2AKO) mice. We also found that pulmonary endothelial cells (mPEC) isolated from female WT mice showed higher expression of A2A receptor than mPEC from male WT mice. mPEC from female WT mice were more sensitive to A2A-mediated pro-angiogenic response, suggesting an ER and A2A crosstalk, which was confirmed using cells isolated from A2AKO. In those female cells, 17ß-estradiol potentiated A2A-mediated cell proliferation, an effect that was inhibited by selective antagonists of estrogen receptors (ER), ERα, and ERß. Therefore, estrogen regulates the expression and/or pro-angiogenic activity of A2A adenosine receptors, likely involving activation of ERα and ERß receptors. Sexual dimorphism in wound healing observed in the A2AKO mice process reinforces the functional crosstalk between ER and A2A receptors.

Deletion of the adenosine A2A receptor increases the survival rate in a mice model of polymicrobial sepsis.

We aim to investigate the role of A2A receptor in peritonitis-related sepsis by injection of a fecal solution (FS) as a model of polymicrobial infection. C57/black J6 wild-type (WT) and A2A-deficient mice (A2AKO) were exposed to sepsis induced by intraperitoneal injection of a FS (FS-induced peritonitis) or instead was injected with saline buffer (Sham). Survival rate and sepsis score were measured up to 48 h. The presence of bacteria in tissue homogenates was analyzed. Telemetry and speckle laser Doppler were used for systemic blood pressure and peripheral blood perfusion analysis, respectively. Histological analysis and identification of active caspase 3 were performed in selected organs, including the liver. The survival rate of A2AKO mice exposed to FS-induced peritonitis was significantly higher, and the sepsis score was lower than their respective WT counterpart. Injection of FS increases (50 to 150 folds) the number of colonies forming units in the liver, kidney, blood, and lung in WT mice, while these effects were significantly attenuated in A2AKO mice exposed to FS-induced peritonitis. A significant reduction in both systolic and diastolic blood pressure, as well as in the peripheral perfusion was observed in WT and A2AKO mice exposed to FS-induced peritonitis. Although, these last effects were significantly attenuated in A2AKO mice. Histological analysis showed a large perivascular infiltration of polymorphonuclear in the liver of WT and A2AKO mice exposed to FS-induced peritonitis, but again, this effect was attenuated in A2AKO mice. Finally, high expression of active caspase 3 was found only in the liver of WT mice exposed to FS-induced peritonitis. The absence of the A2A receptor increases the survival rate in mice exposed to polymicrobial sepsis. This outcome was associated with both hemodynamic compensation and enhanced anti-bacterial response.

Are the Cognitive Alterations Present in Children Born From Preeclamptic Pregnancies the Result of Impaired Angiogenesis? Focus on the Potential Role of the VEGF Family.

Evidence from clinical studies has proposed that children born from preeclamptic women have a higher risk of suffering neurological, psychological, or behavioral alterations. However, to date, the mechanisms behind these outcomes are poorly understood. Here, we speculate that the neurodevelopmental alterations in the children of preeclamptic pregnancies result from impaired angiogenesis. The pro-angiogenic factors vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) are key regulators of both vascular and neurological development, and it has been widely demonstrated that umbilical blood of preeclamptic pregnancies contains high levels of soluble VEGF receptor type 1 (sFlt-1), a decoy receptor of VEGF. As a consequence, this anti-angiogenic state could lead to long-lasting neurological outcomes. In this non-systematic review, we propose that alterations in the circulating concentrations of VEGF, PlGF, and sFlt-1 in preeclamptic pregnancies will affect both fetal cerebrovascular function and neurodevelopment, which in turn may cause cognitive alterations in post-natal life.

Gestational diabetes mellitus is associated with increased pro-migratory activation of vascular endothelial growth factor receptor 2 and reduced expression of vascular endothelial growth factor receptor 1.

Placentas from gestational diabetes mellitus (GDM) are often hypervascularized; however, participation of vascular endothelial growth factor (VEGF) and its receptors in this placental adaptation is unclear. We aimed to test whether changes in phosphorylation of tyrosine 951 or tyrosine 1175 (pY951 or pY1175) of the vascular endothelial growth factor receptor 2 (KDR) are associated with the proangiogenic state observed in placentas from GDM. We obtained placental samples from women with normal pregnancies (n = 24) or GDM (n = 18). We measured the relative expression of markers for endothelial cell number (CD31, CD34), VEGF, vascular endothelial growth factor receptor 1 (Flt-1), KDR, pY951 and pY1175 of KDR in placental homogenate. Immunohistochemistry of placental blood vessels were performed using CD34. Proliferation and migration of human umbilical vein endothelial cells (HUVEC) obtained from normal pregnancy and GDM were determined in absence or presence of conditioned medium (CM) harvested from GDM or normoglycemic HUVEC cultures. GDM was associated with more CD31 and CD34 protein compared to normal pregnancy. High number, but reduced area of placental blood vessels was found in GDM. Reduced Flt-1 levels (mRNA and protein) are associated with reduced KDR mRNA, but higher KDR protein levels in placentas from GDM. No significant changes in Y951-or Y1175-phosphorylation of KDR in placentas from GDM were found. GDM did not alter proliferation of HUVECs, but enhanced migration. Conditioned medium harvested from GDM HUVEC cultures enhanced KDR protein amount, tube formation capacity and cell migration in HUVEC isolated from normoglycemic pregnancies. The data indicate that GDM is associated with reduced expression of Flt-1 but high pro-migratory activation of KDR reflecting a proangiogenic state in GDM.

High plasma adenosine levels in overweight/obese pregnant women.

We aim to investigate whether overweight/obese pregnant women have elevated plasma levels of adenosine associated with increased consumption of high-calorie food. Sixty women were included. They were divided into lean (n = 23 and n = 12) or overweight/obese (n = 7 and n = 18) non-pregnant and pregnant women, respectively. Clinical records and maternal blood samples were collected after informed consent. A self-reported dietary questionnaire was also completed. Plasma adenosine levels were determined with high-performance liquid chromatography. Biochemical parameters, including glucose, total protein, and lipid profile, were determined using standard colorimetric assays. Adenosine levels were higher in pregnant women than in non-pregnant women (18.7 ± 1.6 vs 10.8 ± 1.3 nM/µg protein, respectively, p < 0.0001). Overweight/obese pregnant women (21.9 ± 2.5 nM/µg protein) exhibited higher adenosine levels than lean pregnant (14.5 ± 1.0 nM/µg protein, p = 0.04) or non-pregnant women (11.7 ± 1.5 nM/µg protein, p = 0.0005). Also, pregnant women with elevated weight gain exhibited higher (26.2 ± 3.7 nM/µg protein) adenosine levels than those with adequate weight gain (14.9 ± 1.4 nM/µg protein, p = 0.03). These differences were not statistically significant compared with those of pregnant women with reduced weight gain (17.4 ± 2.1 nM/µg protein, p = 0.053). Body mass index and adenosine only in pregnant women were positively correlated (r = 0.39, p = 0.02). While, polyunsaturated fatty acid (PUFA) consumption was negatively correlated with plasma adenosine levels only in non-pregnant women (r = -0.33, p = 0.03). Pregnancy is associated with high plasma adenosine levels, which are further elevated in pregnant women who are overweight/obese. High PUFA intake might reduce plasma adenosine levels in non-pregnant women.

Pro-angiogenic Role of Insulin: From Physiology to Pathology.

The underlying molecular mechanisms involve in the regulation of the angiogenic process by insulin are not well understood. In this review article, we aim to describe the role of insulin and insulin receptor activation on the control of angiogenesis and how these mechanisms can be deregulated in human diseases. Functional expression of insulin receptors and their signaling pathways has been described on endothelial cells and pericytes, both of the main cells involved in vessel formation and maturation. Consequently, insulin has been shown to regulate endothelial cell migration, proliferation, and in vitro tubular structure formation through binding to its receptors and activation of intracellular phosphorylation cascades. Furthermore, insulin-mediated pro-angiogenic state is potentiated by generation of vascular growth factors, such as the vascular endothelial growth factor, produced by endothelial cells. Additionally, diseases such as insulin resistance, obesity, diabetes, and cancer may be associated with the deregulation of insulin-mediated angiogenesis. Despite this knowledge, the underlying molecular mechanisms need to be elucidated in order to provide new insights into the role of insulin on angiogenesis.

Adenosine A2A receptor regulates expression of vascular endothelial growth factor in feto-placental endothelium from normal and late-onset pre-eclamptic pregnancies.

We aim to investigate whether A2A/nitric oxide-mediated regulation of vascular endothelial growth factor (VEGF) expression is impaired in feto-placental endothelial cells from late-onset pre-eclampsia. Cultures of human umbilical vein endothelial cells (HUVECs) and human placental microvascular endothelial cells (hPMECs) from normal and pre-eclamptic pregnancies were used. Assays by using small interference RNA (siRNA) for A2A were performed, and transfected cells were used for estimation of messenger RNA (mRNA) levels of VEGF, as well as for cell proliferation and angiogenesis in vitro. CGS-21680 (A2A agonist, 24 h) increases HUVEC and hPMEC proliferation in a dose response manner. Furthermore, similar to CGS-21680, the nitric oxide donor, S-nitroso-N-acetyl-penicillamine oxide (SNAP), increased cell proliferation in a dose response manner (logEC50 10-9.2 M). In hPMEC, CGS-21680 increased VEGF protein levels in both normal (∼1.5-fold) and pre-eclamptic pregnancies (∼1.2-fold), an effect blocked by the A2A antagonist, ZM-241385 (10-5 M) and the inhibitor of NO synthase, N ω-nitro-L-arginine methyl ester hydrochloride (L-NAME). Subsequently, SNAP partially recovered cell proliferation and in vitro angiogenesis capacity of cells from normal pregnancies exposed to siRNA for A2A. CGS-21680 also increased (∼1.5-fold) the level of VEGF mRNA in HUVEC from normal pregnancies, but not in pre-eclampsia. Additionally, transfection with siRNA for A2A decrease (∼30 %) the level of mRNA for VEGF in normal pregnancy compared to untransfected cells, an effect partially reversed by co-incubation with SNAP. The A2A-NO-VEGF pathway is present in endothelium from microcirculation and macrocirculation in both normal and pre-eclamptic pregnancies. However, NO signaling pathway seems to be impaired in HUVEC from pre-eclampsia.

Role of Extracellular Vesicles and microRNAs on Dysfunctional Angiogenesis during Preeclamptic Pregnancies.

Preeclampsia is a syndrome characterized by hypertension during pregnancy, which is a leading cause of morbidity and mortality in both mother and newborn in developing countries. Some advances have increased the understanding of pathophysiology of this disease. For example, reduced utero-placental blood flow associated with impaired trophoblast invasion may lead to a hypoxic placenta that releases harmful materials into the maternal and feto-placental circulation and impairs endothelial function. Identification of these harmful materials is one of the hot topics in the literature, since these provide potential biomarkers. Certainty, such knowledge will help us to understand the miscommunication between mother and fetus. In this review we highlight how placental extracellular vesicles and their cargo, such as small RNAs (i.e., microRNAs), might be involved in endothelial dysfunction, and then in the angiogenesis process, during preeclampsia. Currently only a few reports have addressed the potential role of endothelial regulatory miRNA in the impaired angiogenesis in preeclampsia. One of the main limitations in this area is the variability of the analyses performed in the current literature. This includes variability in the size of the particles analyzed, and broad variation in the exosomes considered. The quantity of microRNA targets genes suggest that practically all endothelial cell metabolic functions might be impaired. More studies are required to investigate mechanisms underlying miRNA released from placenta upon endothelial function involved in the angiogenenic process.

Acoustic parameters and salivary IL-6 levels in overweight and obese teachers.

OBJECTIVE: Either obesity or vocal loading task leads to elevation of proinflammatory cytokines such as interleukin 6 (IL-6). However, it is unknown whether vocal parameters after vocal loading are correlated with body mass index (BMI) or IL-6. We hypothesize that vocal loading induces an elevation of acoustic parameters of voice and salivary IL-6 in overweight and obese people. METHODS: A total of 33 schoolteachers without any self-reported voice alterations were invited to participate in this study. Participants were classified according to BMI into normal, overweight, and obese groups. The vocal loading task consisted of loud speech (60-90 minutes) in their classroom. Salivary and voice samples were taken before and after vocal loading. Perceptual and self-reported voice alterations and objective voice analyses were investigated. The relative concentration of salivary IL-6 was estimated by a colorimetric assay. RESULTS: Obese teachers showed a significant elevation in fundamental frequency value after vocal loading. In addition, reduction in harmonics-to-noise ratio (HNR) was observed in teachers with normal weight after vocal loading but not in overweight or obese groups. No significant correlation was observed between BMI and any of the acoustic parameters analyzed or salivary IL-6 levels. Furthermore, teachers who were overweight showed a significant increase in the salivary IL-6 levels after vocal loading. Interestingly, salivary IL-6 levels were positively correlated with HNR value in the overweight group after vocal loading. CONCLUSION: Excessive body weight is related to alterations in fundamental frequency and HNR. In addition, HNR, but not BMI, is associated with salivary IL-6 levels in overweight teachers.