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The lectin-like oxidized low-density-lipoprotein (oxLDL) receptor-1 (LOX-1) has been shown to induce angiotensin II (AngII) type 1 receptor (AT1) activation, contributing to vascular dysfunction. Preeclampsia is a pregnancy complication characterized by vascular dysfunction and increased LOX-1 and AT1 activation; however, whether LOX-1 and AT1 activity contributes to vascular dysfunction in preeclampsia is unknown. We hypothesized that increased oxLDL levels during pregnancy lead to LOX-1 activation and subsequent AT1 activation, resulting in vascular dysfunction. Pregnant wild-type (WT) and transgenic LOX-1 overexpressing (LOX-1tg) mice were fed a control diet (CD) or high-cholesterol diet (HCD, to impair vascular function) between gestational day (GD) 13.5-GD18.5. On GD18.5, AngII-induced vasoconstriction and methylcholine (MCh)-induced endothelium-dependent vasodilation responses were assessed in aortas and uterine arteries. HCD decreased fetal weight and increased circulating oxLDL/cholesterol levels in WT, but not in LOX-1tg mice. HCD did not alter AngII responsiveness or AT1 expression in both vascular beds; however, AngII responsiveness and AT1 expression were lower in aortas from LOX-1tg compared with WT mice. In aortas from WT-CD mice, acute oxLDL exposure induced AT1-mediated vasoconstriction via LOX-1. HCD impaired endothelium-dependent vasodilation and increased superoxide levels in WT aortas, but not uterine arteries. Moreover, in WT-CD mice oxLDL decreased MCh sensitivity in both vascular beds, partially via LOX-1. In summary, HCD impaired pregnancy outcomes and vascular function, and oxLDL-induced LOX-1 activation may contribute to vascular dysfunction via AT1. Our study suggests that LOX-1 could be a potential target to prevent adverse outcomes associated with vascular dysfunction in preeclampsia.

Vascular dysfunction is a hallmark of cardiovascular disease (CVD). Offspring from preeclamptic pregnancies are at risk of CVD later in life. Whether fetal vasculature from preeclamptic pregnancies displays signs of vascular dysfunction (i.e., oxidative/nitrosative stress, endothelial activation) associated with increased expression of lectin-like oxidized LDL receptor-1 (LOX-1) and angiotensin-II type-1 receptor (AT1) is unknown. We demonstrated increased superoxide, nitrotyrosine and ICAM-1 levels in umbilical vein tissues of preeclamptic vs. normal pregnancies; without changes in LOX-1 and AT1 levels. Our findings suggest that the fetal vasculature may be impacted in preeclampsia, which could contribute to an increased risk of offspring CVD.

Insulin resistance plays a key role in cardiovascular complications associated with diabetes mellitus and hypertensive disorders. In states of insulin resistance several circulating factors may contribute to a defective insulin sensitivity in different tissues, including the vasculature. One of these factors influencing the vascular insulin resistance are the extracellular vesicles. The extracellular vesicles include exosomes, microvesicles, and apoptotic bodies which are released to the circulation by different vascular cells. Since the cargo of extracellular vesicles seems to be altered in metabolic complications associated with insulin resistance, these vesicles may be candidates contributing to vascular insulin resistance. Despite the studies linking insulin resistance signalling pathways with the vascular effect of extracellular vesicles, the involvement of these structures in vascular insulin resistance is a phenomenon that remains unclear.

Gestational diabetes mellitus (GDM) is associated with fetoplacental endothelial dysfunction, which may be induced by hyperglycemia. We hypothesized that endothelial exosomes, which are extracellular nanovesicles affecting endothelial function, play a role in the high glucose (HG)-induced endothelial dysfunction. METHODS: Exosomes were isolated from HUVECs incubated with basal glucose (5.5 mmol/L; HUVEC- BG; exo-BG) and from HUVECs incubated with HG for 24 h (25 mmol/L; HUVEC-HG; exo-HG) in exosome-free medium. Exosomes were isolated and characterized by ultracentrifugation, sucrose gradient, electron microscopy, nanotracking analysis and Western blotting. HUVEC-BG and HUVEC-HG were exposed to exo-BG and exo-HG in two different concentrations: 5 μg and 1 μg exosome protein/mL. The exosomal effect on endothelial cell function was determined by wound healing assay, expression of endothelial nitric oxide synthase (eNOS), human cationic amino acid transporter type 1 (hCAT-1), vascular endothelial growth factor (VEGF) and intracellular adhesion molecule type 1 (ICAM-1) by Western blotting, qPCR or flow cytometry. RESULTS: HG increased the exosomal release from HUVECs, endothelial wound healing and expression of phosphorylated (P∼Ser1177)-eNOS, hCAT-1, VEGF and ICAM-1. Exo-HG also increased endothelial cell wound healing, P∼Ser1177-eNOS, hCAT-1 and ICAM-1 expression in HUVEC-BG. Exo-BG reverted the effect of HG on endothelial cell wound healing and hCAT-1 mRNA expression to normal values. DISCUSSION: Our results show that HG may induce endothelial dysfunction in HUVECs and that exosomes from HUVEC-HG mimicked some of the effects of HG. This study contributes to the unraveling of the mechanism by which hyperglycemia affects the fetoplacental vasculature in GDM. 7th European Community Framework Program (grant agreement No. 295185 – EULAMDIMA)

Regulation of blood flow depends on systemic and local release of vasoactive molecules such as insulin and adenosine. These molecules cause vasodilation by activation of plasma membrane receptors at the vascular endothelium. Adenosine activates at least four subtypes of adenosine receptors (A(1)AR, A(2A)AR, A(2B)AR, A(3)AR), of which A(2A)AR and A(2B)AR activation leads to increased cAMP level, generation of nitric oxide, and relaxation of the underlying smooth muscle cell layer. Vasodilation caused by adenosine also depends on plasma membrane hyperpolarization due to either activation of intermediate-conductance Ca2+-activated K+ channels in vascular smooth muscle or activation of ATP-activated K+ channels in the endothelium. Adenosine also causes vasoconstriction via a mechanism involving A(1)AR activation resulting in lower cAMP level and increased thromboxane release. Insulin has also a dual effect causing NO-dependent vasodilation, but also sympathetic activity-and increased endothelin 1 release-dependent vasoconstriction. Interestingly, insulin effects require or are increased by activation or inactivation of adenosine receptors. This is phenomenon described for D-glucose and L-arginine transport where A(2A)AR and A(2B)AR play a major role. Other studies show that A(1)AR activation could reduce insulin release from pancreatic beta-cells. Whether adenosine modulation of insulin biological effect is a phenomenon that depends on co-localization of adenosine receptors and insulin receptors, and adenosine plasma membrane transporters is something still unclear. This review summarizes findings addressing potential involvement of adenosine receptors to modulate insulin effect via insulin receptors with emphasis in the human vasculature. (C) 2016 Elsevier Ltd. All rights reserved.

The human insulin receptor (IR) exists in two isoforms that differ by the absence (IR-A) or the presence (IR-B) of a 12-amino acid segment encoded by exon 11. Both isoforms are functionally distinct regarding their binding affinities and intracellular signalling. However, the underlying mechanisms related to their cellular functions in several tissues are only partially understood. In this review, we summarize the current knowledge in this field regarding the alternative splicing of IR isoform, tissue-specific distribution and signalling both in physiology and disease, with an emphasis on the human placenta in gestational diabetes mellitus (GDM). Furthermore, we discuss the clinical relevance of IR isoforms highlighted by findings that show altered insulin signalling due to differential IR-A and IR-B expression in human placental endothelium in GDM pregnancies. Future research and clinical studies focused on the role of IR isoform signalling might provide novel therapeutic targets for treating GDM to improve the adverse maternal and neonatal outcomes.

Gestational diabetes mellitus (GDM) characterizes by foetoplacental endothelial dysfunction. Human umbilical vein endothelial cells (HUVECs) from women with GDM show increased L-arginine transport via the human cationic amino acid transporter 1 (hCAT-1). Moreover, expression of endothelial nitric oxide synthase (eNOS) and nitric oxide synthesis are increased. Exosomes are increased in maternal plasma from GDM. We evaluated the role of foetoplacental endothelial exosomes on endothelial dysfunction in GDM. Exosomes were isolated from HUVECs from normal (ExN) and GDM (ExGDM) pregnancies. HUVECs were exposed (8h) to ExN or ExGDM and used for wound recovery assay (up to 8h), L-arginine transport, hCAT-1 and eNOS expression and activity, reactive oxygen species (ROS) generation, and 44 and 42kDa mitogen activated protein kinases (p44/42(mapk)) and protein kinase B/Akt (Akt) activation. Wound recovery was slower in GDM compared with normal pregnancies and was recovered by ExN. However, ExGDM delayed wound recovery in cells from normal pregnancies. GDM-increased L-arginine transport, hCAT-1 and eNOS expression and activity, and p44/42(mapk) activation were blocked by ExN, but ExGDM increased these parameters and ROS generation, and reduced eNOS phosphorylation at threonine(495) in cells from normal pregnancies. Inhibition of p44/42(mapk), but not Akt reversed GDM-increased L-arginine uptake. In conclusion foetoplacental endothelial-released exosomes play a role in the maintenance of a GDM phenotype in HUVECs. It is suggested that ExN and ExGDM cargo are different with differential effects in cells from normal or GDM pregnancies. This phenomenon could contribute to the understanding of mechanisms behind foetoplacental endothelial dysfunction in GDM pregnancies.

Introduction: Maternal hypercholesterolemia and hypertriglyceridemia during pregnancy is correlated with fetoplacental endothelial dysfunction and atherosclerotic lesions in fetal arteries. Few studies have reported the distribution of the concentrations of maternal total cholesterol (TCh), lipoprotein cholesterol and triglycerides during pregnancy. Therefore, we determined maternal lipid concentration during pregnancy and established the percentiles over which fetoplacental endothelial dysfunction is observed. Methods: A lipoprotein profile was determined for 249 pregnant Chilean women in each trimester of pregnancy in cross-sectional and longitudinal lipid determination studies. Distribution percentiles for TCh, highe, lowe and very-lowedensity lipoprotein (HDL, LDL, and vLDL, respectively) cholesterol and triglycerides were estimated. The reactivity of human umbilical vein rings to the calcitonin gene-related peptide (0.1e1000 nmol/L, 5 min) and sodium nitroprusside (10 mmol/L, 5 min) was measured (wire myography) in KCl-preconstricted vessels. Results: Maternal lipoproteins and triglyceride concentrations increased over time from preconception to the 3rd trimester of pregnancy. Newborn umbilical blood lipoprotein and triglyceride concentrations were lower than those in maternal circulation. Changes in maternal HDL correlated with newborn HDL concentration; however, no correlation between maternal lipoprotein concentrations and newborn weight was found. Maternal TCh and LDL concentrations were inversely correlated with the maximal dilation, but the >75th percentile of maternal TCh and LDL concentrations (>291 and >169 mg/dL, respectively) correlated with reduced calcitonin gene-related peptide sensitivity of the vein rings. Discussion and conclusion: We identified percentiles for maternal TCh and LDL concentrations over which abnormal endothelium-dependent human fetoplacental vascular response is observed.

New Findings: What is the topic of this review? This review focuses on the effects of insulin therapy on fetoplacental vasculature in gestational diabetes mellitus and the potentiating effects of adenosine on this therapy. What advances does it highlight? This review highlights recent studies exploring a potential functional link between insulin receptors and their dependence on adenosine receptor activation (insulin-adenosine axis) to restore placental endothelial function in gestational diabetes mellitus. Gestational diabetes mellitus (GDM) is a disease that occurs during pregnancy and is associated with maternal and fetal hyperglycaemia. Women with GDM are treated via diet to control their glycaemia; however, a proportion of these patients do not achieve the recommended values of glycaemia and are subjected to insulin therapy until delivery. Even if a diet-treated GDM pregnancy leads to normal maternal and newborn glucose levels, fetoplacental vascular dysfunction remains evident. Thus, control of glycaemia via diet does not prevent GDM-associated fetoplacental vascular and metabolic alterations. We review the available information regarding insulin therapy in the context of its potential consequences for fetoplacental vascular function in GDM. We propose the possibility that insulin therapy to produce normoglycaemia in the mother and newborn may require additional therapeutic measures to restore the normal metabolic condition of the vascular network in GDM. A role for A and A adenosine receptors and insulin receptors A and B as well as a potential functional link in the cell signalling associated with the activation of these receptors is proposed. This possibility could be helpful for the planning of strategies, including adenosine receptor-improved insulin therapy, for the treatment of GDM patients, thereby promoting the wellbeing of the growing fetus, newborn and mother.

Insulin resistance is characteristic of pregnancies where the mother shows metabolic alterations, such as preeclampsia (PE) and gestational diabetes mellitus (GDM), or abnormal maternal conditions such as pregestational maternal obesity (PGMO). Insulin signalling includes activation of insulin receptor substrates 1 and 2 (IRS1/2) as well as Src homology 2 domain-containing transforming protein 1, leading to activation of 44 and 42 kDa mitogen-activated protein kinases and protein kinase B/Akt (Akt) signalling cascades in the human foetoplacental vasculature. PE, GDM, and PGMO are abnormal conditions coursing with reduced insulin signalling, but the possibility of the involvement of similar cell signalling mechanisms is not addressed. This review aimed to determine whether reduced insulin signalling in PE, GDM, and PGMO shares a common mechanism in the human foetoplacental vasculature. Insulin resistance in these pathological conditions results from reduced Akt activation mainly due to inhibition of IRS1/2, likely due to the increased activity of the mammalian target of rapamycin (mTOR) resulting from lower activity of adenosine monophosphate kinase. Thus, a defective signalling via Akt/mTOR in response to insulin is a central and common mechanism of insulin resistance in these diseases of pregnancy. In this review, we summarise the cell signalling mechanisms behind the insulin resistance state in PE, GDM, and PGMO focused in the Akt/mTOR signalling pathway in the human foetoplacental endothelium. Unión Europea Framework Grant Agreement no. 295185–EULAMDIMA

Gestational diabetes mellitus (GDM) is a disease of pregnancy associated with endothelial dysfunction in the foetoplacental vasculature. Foetoplacental endothelial dysfunction is characterized by changes in the l-arginine-adenosine signalling pathway and inflammation. The mechanisms involved in these alterations are suggested to be hyperglycaemia, hyperinsulinemia, and oxidative stress. These conditions increase the release of exosomes, nanovesicles that are generated from diverse cell types, including endothelial cells. Since exosomes can modulate vascular function, they may play an important role in foetoplacental endothelial dysfunction seen in GDM pregnancies. In this review, we summarized current knowledge on the potential role of exosomes in foetoplacental endothelial dysfunction seen in this disease of pregnancy.

Reduced adenosine uptake via human equilibrative nucleoside transporter 1 (hENT1) in human umbilical vein endothelial cells (HUVECs) from gestational diabetes mellitus (GDM) is reversed by insulin by restoring hENT1 expression. Insulin receptors A (IR-A) and B (IR-B) are expressed in HUVECs, and GDM results in higher IR-A mRNA expression vs. cells from normal pregnancies. We studied whether the reversal of GDM effects on transport by insulin depends on restoration of IR-A expression. We specifically measured hENT1 expression [mRNA, protein abundance, SLC29A1 (for hENT1) promoter activity] and activity (adenosine transport kinetics) and the role of IR-A/IR-B expression and signaling [total and phosphorylated 42 and 44 kDa mitogen-activated protein kinases (p44/42(mapk)) and Akt] in IR-A, IR-B, and IR-A/B knockdown HUVECs from normal (n = 33) or GDM (n = 33) pregnancies. GDM increases IR-A/IR-B mRNA expression (1.8-fold) and p44/42(mapk):Akt activity (2.7-fold) ratios. Insulin reversed GDM-reduced hENT1 expression and maximal transport capacity (V(max)/K(m)), and GDM-increased IR-A/IR-B mRNA expression and p44/42(mapk):Akt activity ratios to values in normal pregnancies. Insulin's effect was abolished in IR-A or IR-A/B knockdown cells. Thus, insulin requires normal IR-A expression and p44/42(mapk)/Akt signaling to restore GDM-reduced hENT1 expression and activity in HUVECs. This could be a protective mechanism for the placental macrovascular endothelial dysfunction seen in GDM.

Gestational diabetes mellitus (GDM) is a diseases that alters human placenta macro and microvascular reactivity as a result of endothelial dysfunction. The human placenta is a highly vascularized organ which lacks innervation, so blood flux is governed by locally released vasoactive molecules, including the endogenous nucleoside adenosine and the free radical nitric oxide (NO). Altered adenosine metabolism and uptake by the endothelium leads to increased NO synthesis which then turns-off the expression of genes coding for a family of nucleoside membrane transporters belonging to equilibrative nucleoside transporters, particularly isoforms 1 (hENT1) and 2 (hENT2). This mechanism leads to increased extracellular adenosine and, as a consequence, activation of adenosine receptors to further sustain a tonic activation of NO synthesis. This is a phenomenon that seems operative in the placental macro and microvascular endothelium in GDM. We here summarize the findings available in the literature regarding these mechanisms in the human feto-placental circulation. This phenomenon is altered in the feto-placental vasculature, which could be crucial for understanding GDM deleterious effects in fetal growth and development.

The placenta is a vital organ whose function in diseases of pregnancy is altered, resulting in an abnormal supply of nutrients to the foetus. The lack of placental vasculature homeostasis regulation causes endothelial dysfunction and altered vascular reactivity. The proper distribution of acid- (protons (H(+))) and base-equivalents through the placenta is essential to achieve physiological homeostasis. Several membrane transport mechanisms that control H(+) distribution between the extracellular and intracellular spaces are expressed in the human placenta vascular endothelium and syncytiotrophoblast, including sodium (Na(+))/H(+) exchangers (NHEs). One member of the NHEs family is NHE isoform 1 (NHE1), whose activity results in an alkaline intracellular pH (high intracellular pH (pHi)) and an acidic extracellular pH (pHo). Increased NHE1 expression, maximal transport activity, and turnover are reported in human syncytiotrophoblasts and lymphocytes from patients with diabetes mellitus type I (DMT1), and a positive correlation between NHEs activity and plasma factors, such as that between thrombin and platelet factor 3, has been reported in diabetes mellitus type II (DMT2). However, gestational diabetes mellitus (GDM) could result in a higher sensitivity of the human placenta to acidic pHo. We summarized the findings on pHi and pHo modulation in the human placenta with an emphasis on pregnancies in which the mother diagnosed with diabetes mellitus. A potential role of NHEs, particularly NHE1, is proposed regarding placental dysfunction in DMT1, DMT2, and GDM.

Preeclampsia (PE), gestational diabetes mellitus (GDM), and maternal supraphysiological hypercholesterolaemia (MSPH) are pregnancy-related conditions that cause metabolic disruptions leading to alterations of the mother, fetus and neonate health. These syndromes result in fetoplacental vascular dysfunction, where nitric oxide (NO) plays a crucial role. PE characterizes by abnormal increase in the placental blood pressure and a negative correlation between NO level and fetal weight, suggesting that increased NO level and oxidative stress could be involved. GDM courses with macrosomia along with altered function of the fetal cardiovascular system and fetoplacental vasculature. Even when NO synthesis in the fetoplacental vasculature is increased, NO bioavailability is reduced due to the higher oxidative stress seen in this disease. In MSPH, there is an early development of atherosclerotic lesions in fetal and newborn arteries, altered function of the fetoplacental vasculature, and higher markers of oxidative stress in fetal blood and placenta, thus, vascular alterations related with NO metabolism occur as a consequence of this syndrome. Potential mechanisms of altered NO synthesis and bioavailability result from transcriptional and post-translational NO synthases (NOS) modulation, including phosphorylation/dephosphorylation cycles, coupling/uncoupling of NOS, tetrahydrobiopterin bioavailability, calcium/calmodulin-NOS and caveolin-1-NOS interaction. Additionally, oxidative stress also plays a role in the reduced NO bioavailability. This review summarizes the available information regarding lower NO bioavailability in these pregnancy pathologies. A common NO-dependent mechanism in PE, GDM and MSPH contributing to fetoplacental endothelial dysfunction is described.

Human foetal development and growth in an environment of maternal obesity associates with high risk of cardiovascular disease and adverse neonatal outcome. We studied whether supraphysiological gestational weight gain results in human fetoplacental endothelial dysfunction and altered fetoplacental vascular reactivity.Primary cultures of human umbilical vein endothelial cells (HUVECs) and umbilical vein rings were obtained from pregnant women (112 total of patients recruited, 7 patients dropped out) exhibiting prepregnancy normal weight that ended with a physiological (pGWG (n=67), total weight gain 11.5-16 kg, rates of weight gain ≤0.42 kg per week) or supraphysiological (spGWG (n=38), total weight gain16 kg, rates of weight gain0.42 kg per week) gestational weight gain (reference values from US Institute of Medicine guidelines). Vascular reactivity to insulin (0.1-1000 nmol l(-1), 5 min) in KCl-preconstricted vein rings was measured using a wire myograph. Protein levels of human equilibrative nucleoside transporter 1 (hENT1), total and Ser(1177)- or Thr(495)-phosphorylated endothelial nitric oxide synthase (eNOS) were detected by western blot or immunofluorescence, and adenosine transport (0-250 μmol l(-1) adenosine, 2 μCi ml(-1) [(3)H]adenosine, 20 s, 25 °C) was measured in the presence or absence of 1 μmol l(-1) nitrobenzylthioinosine (hENT1 inhibitor) or 10 μmol l(-1) chlorpromazine (CPZ, endocytosis inhibitor) in HUVECs.spGWG associates with reduced NOS activity-dependent dilation of vein rings (P=0.001), lower eNOS expression and higher Thr(495) (P=0.044), but unaltered Ser(1177)eNOS phosphorylation. hENT1-adenosine maximal transport activity was reduced (P=0.041), but the expression was increased (P=0.001) in HUVECs from this group. CPZ increased hENT1-adenosine transport (P=0.040) and hENT1 plasma membrane accumulation only in cells from pGWG.spGWG in women with a normal prepregnancy weight causes lower fetoplacental vascular reactivity owing to the downregulation of eNOS activity and adenosine transport in HUVECs. Maternal spGWG is a detrimental condition for human fetoplacental endothelial function and reducing these alterations could result in a better neonate outcome.

Vascular tone is controlled by the L-arginine/nitric oxide (NO) pathway, and NO bioavailability is strongly affected by hyperglycaemia-induced oxidative stress. Insulin leads to high expression and activity of human cationic amino acid transporter 1 (hCAT-1), NO synthesis and vasodilation; thus, a protective role of insulin on high D-glucose–alterations in endothelial function is likely. Vascular reactivity to U46619 (thromboxane A2 mimetic) and calcitonin gene related peptide (CGRP) was measured in KCl preconstricted human umbilical vein rings (wire myography) incubated in normal (5 mmol/L) or high (25 mmol/L) D-glucose. hCAT-1, endothelial NO synthase (eNOS), 42 and 44 kDa mitogen-activated protein kinases (p42/44mapk), protein kinase B/Akt (Akt) expression and activity were determined by western blotting and qRT-PCR, tetrahydrobiopterin (BH4) level was determined by HPLC, and L-arginine transport (0–1000 μmol/L) was measured in response to 5–25 mmol/L D-glucose (0–36 hours) in passage 2 human umbilical vein endothelial cells (HUVECs). Assays were in the absence or presence of insulin and/or apocynin (nicotinamide adenine dinucleotide phosphate-oxidase [NADPH oxidase] inhibitor), tempol or Mn(III)TMPyP (SOD mimetics). High D-glucose increased hCAT-1 expression and activity, which was biphasic (peaks: 6 and 24 hours of incubation). High D-glucose–increased maximal transport velocity was blocked by insulin and correlated with lower hCAT-1 expression and SLC7A1 gene promoter activity. High D-glucose–increased transport parallels higher reactive oxygen species (ROS) and superoxide anion (O2 •–) generation, and increased U46619-contraction and reduced CGRP-dilation of vein rings. Insulin and apocynin attenuate ROS and O2 •– generation, and restored vascular reactivity to U46619 and CGRP. Insulin, but not apocynin or tempol reversed high D-glucose–increased NO synthesis; however, tempol and Mn(III)TMPyP reversed the high D-glucose–reduced BH4 level. Insulin and tempol blocked the high D-glucose–increased p42/44mapk phosphorylation. Vascular dysfunction caused by high D-glucose is likely attenuated by insulin through the L-arginine/NO and O2 •–/NADPH oxidase pathways. These findings are of interest for better understanding vascular dysfunction in states of foetal insulin resistance and hyperglycaemia.

Introduction Preeclampsia is associated with impaired placental vasodilation and reduced endothelial nitric oxide synthase (eNOS) activity in the foetoplacental circulation. Adenosine and insulin stimulate vasodilation in endothelial cells, and this activity is mediated by adenosine receptor activation in uncomplicated pregnancies; however, this activity has yet to be examined in preeclampsia. Early onset preeclampsia is associated with severe placental vasculature alterations that lead to altered foetus growth and development, but whether late-onset preeclampsia (LOPE) alters foetoplacental vascular function is unknown. Methods Vascular reactivity to insulin (0.1–1000 nmol/L, 5 min) and adenosine (1 mmol/L, 5 min) was measured in KCl-preconstricted human umbilical vein rings from normal and LOPE pregnancies using a wire myograph. The protein levels of human cationic amino acid transporter 1 (hCAT-1), adenosine receptor subtypes, total and Ser 1177 -or Thr 495 -phosphorylated eNOS were detected via Western blot, and l-arginine transport (0–1000 μmol/L l-arginine, 3 μCi/mL L-[ 3 H]arginine, 20 s, 37 °C) was measured in the presence or absence of insulin and adenosine receptor agonists or antagonists in human umbilical vein endothelial cells (HUVECs) from normal and LOPE pregnancies. Results LOPE increased the maximal l-arginine transport capacity and hCAT-1 and eNOS expression and activity compared with normal conditions. The A 2A adenosine receptor (A 2A AR) antagonist ZM-241385 blocked these effects of LOPE. Insulin-mediated umbilical vein ring relaxation was lower in LOPE pregnancies than in normal pregnancies and was restored using the A 2A AR antagonist. Discussion and conclusions The reduced foetoplacental vascular response to insulin may result from A 2A AR activation in LOPE pregnancies.

Objective— Human pregnancy that courses with maternal supraphysiological hypercholesterolemia (MSPH) correlates with atherosclerotic lesions in fetal arteries. It is known that hypercholesterolemia associates with endothelial dysfunction in adults, a phenomenon where nitric oxide (NO) and arginase are involved. However, nothing is reported on potential alterations in the fetoplacental endothelial function in MSPH. The aim of this study was to determine whether MSPH alters fetal vascular reactivity via endothelial arginase/urea and l -arginine transport/NO signaling pathways. Approach and Results— Total cholesterol l -citrulline synthesis from l -arginine, eNOS phosphorylation/dephosphorylation), but increased arginase activity and arginase II protein abundance. Arginase inhibition increased eNOS activity and l -arginine transport capacity without altering human cationic amino acid transporter 1 or human cationic amino acid transporter 2A/B protein abundance in maternal physiological hypercholesterolemia and MSPH. Conclusions— MSPH is a pathophysiological condition altering umbilical vein reactivity because of fetal endothelial dysfunction associated with arginase and eNOS signaling imbalance. We speculate that elevated maternal circulating cholesterol is a factor leading to fetal endothelial dysfunction, which could have serious consequences to the growing fetus.

Microvascular and macrovascular endothelial function maintains vascular reactivity. Several diseases alter endothelial function, including hypertension, obesity, and diabetes mellitus. In addition, micro- and macrovascular endothelial dysfunction is documented in GDM with serious consequences for the growing fetus. Increased l-arginine uptake via hCAT-1 and NO synthesis by eNOS is associated with GDM. These alterations are paralleled by activation of purinergic receptors and increased umbilical vein, but not arteries blood adenosine accumulation. GDM associates with NO-reduced adenosine uptake in placental endothelium, suggested to maintain and/or facilitate insulin vasodilation likely increasing hCAT-1 and eNOS expression and activity. It is proposed that increased umbilical vein blood adenosine concentration in GDM reflects a defective metabolic state of human placenta. In addition, insulin recovers GDM-alterations in hCAT-1 and eNOS in human micro- and macrovascular endothelium, and its biological actions depend on preferential activation of insulin receptors A and B restoring a normal-like from a GDM-like phenotype. We summarized existing evidence for a potential role of insulin/adenosine/micro- and macrovascular endothelial dysfunction in GDM. These mechanisms could be crucial for a better management of the mother, fetus and newborn in GDM pregnancies.

Introduction Placentas from both early-onset (EOPE) and late-onset pre-eclampsia (LOPE) exhibit signs of underperfusion, which in turn, may be associated with altered angiogenesis. Tyrosine 951 (Y951) and Y1175 phosphorylation of the vascular endothelial growth factor receptor 2 (VEGFR2) induced by VEGF triggers the angiogenesis process. Endothelial markers such as CD31 and CD34 have been used for estimating angiogenic processes in several tissues, including placenta. We asked whether vascular density in placental villi was related to Y951/Y1175 phosphorylation of VEGFR2 in LOPE or EOPE. Methods We obtained placental samples from women with normal pregnancies ( n = 22), LOPE ( n = 13), EOPE ( n = 15) and preterm deliveries ( n = 10). Slices from placental tissue were used for CD31 immunostaining. We estimated the expression of CD31, CD34, VEGF, and VEGFR2 by western blot and quantitative PCR. Y951 phosphorylation of VEGFR2 was estimated by western blot, whereas Y1175 phosphorylation was analyzed by ELISA. Results Vessel density in terminal villi and CD31 and CD34 protein abundance were increased in LOPE and EOPE compared to normal pregnancy. However, mRNA levels for CD31 and CD34 were lower in LOPE than in normal pregnancy and VEGF mRNA was higher in EOPE. VEGFR2 protein concentration was not different among the studied groups. Y951 and Y1175 phosphorylation of VEGFR2 was higher in LOPE than in the normotensive group, but only Y951 exhibited greater phosphorylation in EOPE compared to normal pregnancy. Discussion Changes in vessel formation in the pre-eclamptic placenta are controversial. Our study suggests a pro-angiogenic state in both LOPE and EOPE. These changes are however, associated with differential expression of endothelial markers and VEGFR2 activation. Conclusion There is evidence of increased placental angiogenesis in LOPE and EOPE that is associated with differential activation of VEGFR2.

Signal transducers and activators of transcription 3 (Stat3) has been identified as an important signal transducer in the invasive phenotype of the trophoblasts cells in in vitro studies. However, the in situ distribution and patterns of expression of this molecule in trophoblast cells during the development of the placenta are still under-elucidated. Mice uteri of gestational ages between 7 and 14 days of pregnancy (dop) were fixed in methacarn and processed with immunoperoxidase techniques for detection of Stat3 and its phosphorylation at serine (p-ser727) residues, as well as the suppressor of cytokine signaling 3 (Socs3) expression. Stat3 was observed at 7 through 9 dop in both the antimesometrial and mesometrial deciduas, while continued immunoreactivity between 10 and 13 dop was seen only in the mesometrial decidua. In the placenta, Stat3 was detected in the cytotrophoblast cells of labyrinth and giant trophoblast cells between 10 and 14 dop. Immunoreactivity for Stat3 was also seen in trophoblast cells surrounding the maternal blood vessels. On days 10 and 11 of pregnancy, p-ser727 was detectable in the mesometrial decidua and in giant trophoblasts, while during 12-14 dop in the spongiotrophoblast region. In addition, Socs3 was immunodetected in maternal and placental tissues, principally in the giant trophoblast cells during the whole period of the study. The present in situ study shows the distribution of Stat3, its serine activation and Socs3 in different maternal and fetal compartments during murine placental development, thus further supporting the idea that they play a role during physiological placentation in mice.