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Intrauterine growth restriction (IUGR) is a perinatal condition affecting foetal growth, with under the 10th percentile of the weight curve expected for gestational age. This condition has been associated with higher cardiovascular and metabolic risk and post-natal obesity. There are also major changes in placental function, and particularly in a key molecule in this regulation, nitric oxide. The synthesis of nitric oxide has numerous control mechanisms and competition with arginase for their common substrate, the amino acid L-arginine. This competition is reflected in various vascular diseases and particularly in the endothelium of the umbilical vessels of babies with IUGR. Along with this, there is regulation at the epigenetic level, where methylation in specific regions of some gene promoters, such as the nitric oxide synthase, regulating their expression. It is currently of great interest to understand the mechanisms by which diseases such as IUGR may be conditioned, particularly by maternal nutritional and metabolic conditions, and epigenetic mechanisms that could eventually be modifiable, and thus a focus of interest for health interventions., (Copyright © 2016 Sociedad Chilena de Pediatría. Publicado por Elsevier España, S.L.U. All rights reserved.)

Placental vascular tone is critically influenced by nitric oxide (NO) derived from endothelial NO synthase (eNOS) activity. Placental vessels from pregnancies complicated with intrauterine growth restriction present altered NOS-dependent vasodilation. Arginase-2 competes with eNOS for l-arginine and counteracts the NOS-dependent relaxation in umbilical vessels from normal pregnancies. However there is no data regarding the contribution of arginase activity on the impaired endothelial function in IUGR placenta. We studied whether arginase-2 participates in IUGR-related placental vascular dysfunction counteracting eNOS-dependent relaxation, and the regulation of arginase-2 and eNOS expression in endothelial cells from IUGR umbilical arteries (HUAEC) and veins (HUVEC). In IUGR-derived umbilical arteries (UA) and veins (UV), and chorionic arteries (CA), NOS-dependent vasoactive response in the presence and absence of BEC (arginase inhibitor) was studied. Protein levels of eNOS (total and Ser(1177)-P-eNOS), arginase-2 and arginase activity were determined in IUGR HUAEC and HUVEC. In IUGR vessels eNOS-dependent relaxation was reduced, being improved by BEC. This effect was higher in arteries than veins, and in chorionic compared with umbilical vessels. In cultured IUGR endothelial cells, arginase-2 protein expression and activity were increased in HUVEC, without changes in HUAEC. In IUGR-derived endothelium there was a generalized reduction in the in vitro eNOS activation (Ser(1177)-P-eNOS/eNOS), and therefore a decreased eNOS/arginase activity ratio. Here we provide ex vivo and in vitro evidence for a vascular role of arginase throughout placental vasculature, negatively controlling NOS activity. This effect seems to be crucial in the pathophysiology of endothelial dysfunction present in IUGR feto-placental vessels., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Gestational diabetes mellitus (GDM) courses with increased fetal plasma adenosine concentration and reduced adenosine transport in placental macrovascular endothelium. Since insulin modulates human equilibrative nucleoside transporters (hENTs) expression/activity, we hypothesize that GDM will alter hENT2-mediated transport in human placental microvascular endothelium (hPMEC), and that insulin will restore GDM to a normal phenotype involving insulin receptors A (IR-A) and B (IR-B). GDM effect on hENTs expression and transport activity, and IR-A/IR-B expression and associated cell signalling cascades (p42/44 mitogen-activated protein kinases (p42/44(mapk)) and Akt) role in hPMEC primary cultures was assayed. GDM associates with elevated umbilical whole and vein, but not arteries blood adenosine, and reduced hENTs adenosine transport and expression. IR-A/IR-B mRNA expression and p42/44(mapk)/Akt ratios ('metabolic phenotype') were lower in GDM. Insulin reversed GDM-reduced hENT2 expression/activity, IR-A/IR-B mRNA expression and p42/44(mapk)/Akt ratios to normal pregnancies ('mitogenic phenotype'). It is suggested that insulin effects required IR-A and IR-B expression leading to differential modulation of signalling pathways restoring GDM-metabolic to a normal-mitogenic like phenotype. Insulin could be acting as protecting factor for placental microvascular endothelial dysfunction in GDM.

Nitric oxide (NO) is one of the most pleiotropic signaling molecules at systemic and cellular levels, participating in vascular tone regulation, cellular respiration, proliferation, apoptosis and gene expression. Indeed NO actively participates in trophoblast invasion, placental development and represents the main vasodilator in this tissue. Despite the large number of studies addressing the role of NO in the placenta, its participation in placental vascular development and the effect of altered levels of NO on placental function remains to be clarified. This review draws a time-line of the participation of NO throughout placental vascular development, from the differentiation of vascular precursors to the consolidation of vascular function are considered. The influence of NO on cell types involved in the origin of the placental vasculature and the expression and function of the nitric oxide synthases (NOS) throughout pregnancy are described. The developmental processes involved in the placental vascular bed are considered, such as the participation of NO in placental vasculogenesis and angiogenesis through VEGF and Angiopoietin signaling molecules. The role of NO in vascular function once the placental vascular tree has developed, in normal pregnancy as well as in pregnancy-related diseases, is then discussed., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Human endothelial dysfunction is a common feature in many diseases of pregnancy, such as gestational diabetes (GD). Metabolic changes include abnormal synthesis of nitric oxide (NO) and abnormal membrane transport of l-arginine and adenosine in primary cultures of human umbilical vein (HUVEC, macrovascular) and placental microvillus (hPMEC, microvascular) endothelial cells. These alterations are associated with modifications in the expression and activity of endothelial (eNOS) and inducible (iNOS) NO synthases, respectively, an effect that is maintained at least up to passage 5 in culture. HUVEC and hPMEC exhibit expression and activity of the human cationic amino acid transporter 1 (hCAT-1), equilibrative nucleoside transporters 1 (hENT1) and hENT2, as well as the corresponding SLC7A1, SLC29A1 and SLC29A2 gene promoter activities. Altered gene expression results from increased NO level, protein kinase C, mitogen-activated protein kinases, and hCHOP-C/EBPα transcription factor activation. Reduced ENT-mediated adenosine transport in GD is associated with stimulation of the l-arginine/NO pathway, and mainly due to reduced expression and activity of hENT1. In addition, hENT2 activity seems able to restore the reduced adenosine transport in GD. Additionally, insulin exerts a differential modulation of endothelial cells from macrocirculation compared with microcirculation, possibly due to expression of different insulin receptor isoforms. It is suggested that a common functional characteristic leading to changes in the bioavailability of adenosine and metabolism of l-arginine is evidenced by human fetal micro and macrovascular endothelium in GD., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Gestational diabetes mellitus (GDM) and obesity in pregnancy (OP) are pathological conditions associated with placenta vascular dysfunction coursing with metabolic changes at the fetoplacental microvascular and macrovascular endothelium. These alterations are seen as abnormal expression and activity of the cationic amino acid transporters and endothelial nitric oxide synthase isoform, that is, the "endothelial L-arginine/nitric oxide signalling pathway." Several studies suggest that the endogenous nucleoside adenosine along with insulin, and potentially arginases, are factors involved in GDM-, but much less information regards their role in OP-associated placental vascular alterations. There is convincing evidence that GDM and OP prone placental endothelium to an "altered metabolic state" leading to fetal programming evidenced at birth, a phenomenon associated with future development of chronic diseases. In this paper it is suggested that this pathological state could be considered as a metabolic marker that could predict occurrence of diseases in adulthood, such as cardiovascular disease, obesity, diabetes mellitus (including gestational diabetes), and metabolic syndrome., (Copyright © 2011 Andrea Leiva et al.)

Pre-eclampsia is associated with elevated maternal blood pressure and proteinuria, altered fetal growth, and increased plasma adenosine concentration in the mother and the fetus. Human equilibrative nucleoside transporters 1 (hENT1) and hENT2 are crucial to maintain physiological plasma levels of adenosine, thus modulating its several biological effects through adenosine receptor activation. However, it is unknown whether hENTs and adenosine receptors are expressed in human placental microvascular endothelium (hPMEC). To assay whether the increased fetal plasma adenosine concentration in pre-eclampsia results from altered hENT-mediated transport, and the potential involvement of adenosine receptors in this phenomenon, we investigated hENTs and A2A and A2B adenosine receptors expression and function in hPMEC. Cells were isolated and cultured from normal pregnancies (n=17) or pre-eclampsia with adequate-for-gestational age fetuses (n=7). hENT1, hENT2, A2A and A2B adenosine receptors were expressed and functional in hPMEC. Extracellular adenosine concentration was higher (4-fold) in pre-eclampsia versus normal pregnancies. hPMEC from pre-eclampsia exhibit increased total transport (hENT1+hENT2), and maximal velocity (Vmax) for hENT2- (2-fold), but reduced Vmax for hENT1-mediated adenosine transport (75%), with no changes in apparent Km. hENT2 expression was increased (4.5-fold), but hENT1 protein abundance was reduced (80%) in pre-eclampsia. Equally, A2A expression was reduced (50-80%) in pre-eclampsia. CGS-21680 (A2A agonist) did not alter hENTs expression or activity, but ZM-241385 (A2A antagonist) blocked pre-eclampsia effects and increased hENT1-mediated transport in normal pregnancies. Thus, A2B adenosine receptors may differentially modulate hENTs in hPMEC, which could be a mechanism attempting to re-establish physiological extracellular adenosine levels in pre-eclampsia.

Abstract: The main vasodilator in the placenta is nitric oxide (NO), which is synthesized by endothelial NO synthase (eNOS). Arginase-2 competes with eNOS for l-arginine, and its activity has been related with vascular dysfunction. Recently, we showed that hypoxia induces arginase-2, and decreases eNOS activity in human umbilical vein endothelial cells (HUVEC). However there is evidence that vascular responses to hypoxia are not similar throughout the placental vascular tree. We studied whether arginase-2 plays a role controlling vascular tone in human umbilical vessels, and the changes in the expression of arginase-2 and eNOS proteins by hypoxia in endothelial cells from umbilical arteries (HUAEC) and veins (HUVEC). In isolated umbilical vessels the presence of eNOS and arginase-2 was determined in the endothelium, and the NO-dependent vasoactive responses in the presence and absence of S-(2-boronoethyl)-L-cysteine (BEC, arginase inhibitor) were studied. Additionally, HUAEC and HUVEC were exposed (0–24 h) to hypoxia (2% O2) or normoxia (5% O2), and protein levels of eNOS (total and phosphorylated at serine-1177) and arginase-2 were determined. In umbilical arteries and veins arginase-2 and eNOS were detected mainly at the endothelium. BEC induced a higher concentration-dependent relaxation in umbilical arteries than veins, and these responses were NOS-dependent. In HUAEC exposed to hypoxia there were no changes in eNOS and arginase-2 levels, however there was a significant increase of p-eNOS. In contrast, HUVEC showed an increase in arginase-2 and a reduction of p-eNOS in response to hypoxia. These results show that arginases have a vascular role in placental vessels counteracting the NOS-dependent relaxation, which is differentially regulated in placental artery and vein endothelial cells. [Copyright &y& Elsevier]

Abstract: Hypoxia relates with altered placental vasodilation, and in isolated endothelial cells, it reduces activity of the endothelial nitric oxide synthase (eNOS) and l-arginine transport. It has been reported that arginase-2 expression, an alternative pathway for l-arginine metabolism, is increased in adult endothelial cells exposed to hypoxia as well as in pre-eclamptic placentae. We studied in human umbilical vein endothelial cells (HUVEC) whether hypoxia-reduced NO synthesis results from increased arginase-mediated l-arginine metabolism and changes in subcellular localization of eNOS and arginase-2. In HUVEC exposed (24 h) to 5% (normoxia) or 2% (hypoxia) oxygen, l-arginine transport kinetics, arginase activity (urea assay), and NO synthase (NOS) activity (l-citrulline assay) were determined. Arginase-1, arginase-2 and eNOS expression were determined by RT-PCR and Western blot. Subcellular localization of arginase-2 and eNOS were studied using confocal microscopy and indirect immunofluorescence. Experiments were done in absence or presence of S-(2-boronoethyl)-l-cysteine-HCl (BEC, arginase inhibitor) or N G-nitro-l-arginine methyl ester (l-NAME). Hypoxia-induced reduction in eNOS activity was associated with a reduction in eNOS phosphorylation at Serine-1177 and increased phosphorylation at Threonine-495. This was paralleled with an induction in arginase-2 expression and activity, and decreased l-arginine transport. In hypoxia the arginase inhibition, restored NO synthesis and l-arginine transport, without changes in the eNOS post-translational modification status. Hypoxia increased arginase-2/eNOS colocalization, and eNOS redistribution to the cell periphery. Altogether these data reinforce the thought that eNOS cell location, post-translational modification and substrate availability are important mechanisms regulating eNOS activity. If these mechanisms occur in pregnancy diseases where feto-placental oxygen levels are reduced remains to be clarified. [Copyright &y& Elsevier]

Abstract: Pre-eclampsia is associated with elevated maternal blood pressure and proteinuria, altered fetal growth, and increased plasma adenosine concentration in the mother and the fetus. Human equilibrative nucleoside transporters 1 (hENT1) and hENT2 are crucial to maintain physiological plasma levels of adenosine, thus modulating its several biological effects through adenosine receptor activation. However, it is unknown whether hENTs and adenosine receptors are expressed in human placental microvascular endothelium (hPMEC). To assay whether the increased fetal plasma adenosine concentration in pre-eclampsia results from altered hENT-mediated transport, and the potential involvement of adenosine receptors in this phenomenon, we investigated hENTs and A2A and A2B adenosine receptors expression and function in hPMEC. Cells were isolated and cultured from normal pregnancies (n =17) or pre-eclampsia with adequate-for-gestational age fetuses (n =7). hENT1, hENT2, A2A and A2B adenosine receptors were expressed and functional in hPMEC. Extracellular adenosine concentration was higher (4-fold) in pre-eclampsia versus normal pregnancies. hPMEC from pre-eclampsia exhibit increased total transport (hENT1+hENT2), and maximal velocity (V max) for hENT2- (2-fold), but reduced V max for hENT1-mediated adenosine transport (75%), with no changes in apparent K m. hENT2 expression was increased (4.5-fold), but hENT1 protein abundance was reduced (80%) in pre-eclampsia. Equally, A2A expression was reduced (50–80%) in pre-eclampsia. CGS-21680 (A2A agonist) did not alter hENTs expression or activity, but ZM-241385 (A2A antagonist) blocked pre-eclampsia effects and increased hENT1-mediated transport in normal pregnancies. Thus, A2B adenosine receptors may differentially modulate hENTs in hPMEC, which could be a mechanism attempting to re-establish physiological extracellular adenosine levels in pre-eclampsia. [Copyright &y& Elsevier]

Maternal pregestational obesity is a well-known risk factor for offspring obesity, metabolic syndrome, cardiovascular disease and type 2 diabetes. The mechanisms by which maternal obesity can induce alterations in fetal and later neonatal metabolism are not fully elucidated due to its complexity and multifactorial causes. Two adipokines, leptin and adiponectin, are involved in fetal and postnatal growth trajectories, and both are altered in women with pregestational obesity. The placenta synthesizes leptin, which goes mainly to the maternal circulation and in lesser amount to the developing fetus. Maternal pregestational obesity and hyperleptinemia are associated with placental dysfunction and changes in nutrient transporters which directly affect fetal growth and development. By the other side, the embryo can produce its own leptin from early in development, which is associated to fetal weight and adiposity. Adiponectin, an insulin-sensitizing adipokine, is downregulated in maternal obesity. High molecular weight (HMW) adiponectin is the most abundant form and with most biological actions. In maternal obesity lower total and HMW adiponectin levels have been described in the mother, paralleled with high levels in the umbilical cord. Several studies have found that cord blood adiponectin levels are related with postnatal growth trajectories, and it has been suggested that low adiponectin levels in women with pregestational obesity enhance placental insulin sensitivity and activation of placental amino acid transport systems, supporting fetal overgrowth. The possible mechanisms by which maternal pregestational obesity, focusing in the actions of leptin and adiponectin, affects the fetal development and postnatal growth trajectories in their offspring are discussed. [ABSTRACT FROM AUTHOR]