Paola Casanello

Role of nitric oxide in placental vascular development and function

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.

Intrauterine Growth Retardation Is Associated With Reduced Activity and Expression of the Cationic Amino Acid Transport Systems y+/hCAT-1 and y+/hCAT-2B and Lower Activity of Nitric Oxide Synthase in Human Umbilical Vein Endothelial Cells

Intrauterine growth retardation (IUGR) is associated with vascular complications leading to hypoxia and abnormal fetal development. The effect of IUGR on l-arginine transport and nitric oxide (NO) synthesis was investigated in cultures of human umbilical vein endothelial cells (HUVECs). IUGR was associated with membrane depolarization and reduced l-arginine transport (Vmax= 5.8±0.2 versus 3.3±0.1 pmol/&mgr;g protein per minute), with no significant changes in transport affinity (Km=159±15 versus 137±14 &mgr;mol/L). l-Arginine transport was trans-stimulated (8- to 9-fold) in cells from normal and IUGR pregnancies. IUGR was associated with reduced production of l-[3H]citrulline from l-[3H] arginine, lower nitrite and intracellular l-arginine, l-citrulline, and cGMP. IUGR decreased hCAT-1 and hCAT-2B mRNA, and increased eNOS mRNA and protein levels. IUGR-associated inhibition of l-arginine transport and NO synthesis, and membrane depolarization were reversed by the NO donor S-nitroso-N-acetyl-l,d-penicillamine. In summary, endothelium from fetuses with IUGR exhibit altered l-arginine transport and NO synthesis (l-arginine/NO pathway), reduced expression and activity of hCAT-1 and hCAT-2B and reduced eNOS activity. Alterations in l-arginine/NO pathway could be critical for the physiological processes involved in the etiology of IUGR in human pregnancies.

Role of adenosine transport in gestational diabetes-induced L-arginine transport and nitric oxide synthesis in human umbilical vein endothelium

Gestational diabetes is associated with increased l‐arginine transport and nitric oxide (NO) synthesis, and reduced adenosine transport in human umbilical vein endothelial cells (HUVEC). Adenosine increases endothelial l‐arginine/NO pathway via A2 purinoceptors in HUVEC from normal pregnancies. It is unknown whether the effect of gestational diabetes is associated with activation of these purinoceptors or altered expression of human cationic amino acid transporter 1 (hCAT‐1) or human equilibrative nucleoside transporter 1 (hENT1), or endothelial NO synthase (eNOS) in HUVEC. Cells were isolated from normal or gestational diabetic pregnancies and cultured up to passage 2. Gestational diabetes increased hCAT‐1 mRNA expression (2.4‐fold) and activity, eNOS mRNA (2.3‐fold), protein level (2.1‐fold), and phosphorylation (3.8‐fold), but reduced hENT1 mRNA expression (32%) and activity. Gestational diabetes increased extracellular adenosine (2.7 μm), and intracellular l‐arginine (1.9 mm) and l‐citrulline (0.7 mm) levels compared with normal cells (0.05 μm, 0.89 mm, 0.35 mm, respectively). Incubation of HUVEC from normal pregnancies with 1 μm nitrobenzylthioinosine (NBMPR) mimicked the effect of gestational diabetes, but NBMPR was ineffective in diabetic cells. Gestational diabetes and NBMPR effects involved eNOS, PKC and p42/44mapk activation, and were blocked by the A2a purinoceptor antagonist ZM‐241385. Thus, gestational diabetes increases the l‐arginine/NO pathway involving activation of mitogen‐activated protein (MAP) kinases, protein kinase C (PKC) and NO cell signalling cascades following activation of A2a purinoceptors by extracellular adenosine. A functional relationship is proposed between adenosine transport and modulation of l‐arginine transport and NO synthesis in HUVEC, which could be determinant in regulating vascular reactivity in diabetes mellitus.

Review: Differential placental macrovascular and microvascular endothelial dysfunction in gestational diabetes

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.

Insulin Restores Gestational Diabetes Mellitus–Reduced Adenosine Transport Involving Differential Expression of Insulin Receptor Isoforms in Human Umbilical Vein Endothelium

OBJECTIVE To determine whether insulin reverses gestational diabetes mellitus (GDM)–reduced expression and activity of human equilibrative nucleoside transporters 1 (hENT1) in human umbilical vein endothelium cells (HUVECs). RESEARCH DESIGN AND METHODS Primary cultured HUVECs from full-term normal (n = 44) and diet-treated GDM (n = 44) pregnancies were used. Insulin effect was assayed on hENT1 expression (protein, mRNA, SLC29A1 promoter activity) and activity (initial rates of adenosine transport) as well as endothelial nitric oxide (NO) synthase activity (serine1177 phosphorylation, l-citrulline formation). Adenosine concentration in culture medium and umbilical vein blood (high-performance liquid chromatography) as well as insulin receptor A and B expression (quantitative PCR) were determined. Reactivity of umbilical vein rings to adenosine and insulin was assayed by wire myography. Experiments were in the absence or presence of l-NG-nitro-l-arginine methyl ester (l-NAME; NO synthase inhibitor) or ZM-241385 (an A2A-adenosine receptor antagonist). RESULTS Umbilical vein blood adenosine concentration was higher, and the adenosine- and insulin-induced NO/endothelium-dependent umbilical vein relaxation was lower in GDM. Cells from GDM exhibited increased insulin receptor A isoform expression in addition to the reported NO–dependent inhibition of hENT1-adenosine transport and SLC29A1 reporter repression, and increased extracellular concentration of adenosine and NO synthase activity. Insulin reversed all these parameters to values in normal pregnancies, an effect blocked by ZM-241385 and l-NAME. CONCLUSIONS GDM and normal pregnancy HUVEC phenotypes are differentially responsive to insulin, a phenomenon where insulin acts as protecting factor for endothelial dysfunction characteristic of this syndrome. Abnormal adenosine plasma levels, and potentially A2A-adenosine receptors and insulin receptor A, will play crucial roles in this phenomenon in GDM.

Equilibrative Nucleoside Transporter 1 Expression Is Downregulated by Hypoxia in Human Umbilical Vein Endothelium

Reduced oxygen level (hypoxia) induces endothelial dysfunction and release of the endogenous nucleoside adenosine. Human umbilical vein endothelium (HUVEC) function in an environment with 3% to 5% O2 and exhibit efficient adenosine membrane transport via human equilibrative nucleoside transporters 1 (hENT1). We studied whether adenosine transport and hENT1 expression are altered by hypoxia in HUVEC. Hypoxia (0 to 24 hours, 2% and 1% O2) reduced maximal hENT1-adenosine transport velocity (Vmax) and maximal nitrobenzylthionosine (NBMPR, a high-affinity hENT1 protein ligand) binding, but increased extracellular adenosine concentration. Hypoxia also reduced hENT1 protein and mRNA levels, effects unaltered by N&ohgr;-nitro-l-arginine methyl ester (l-NAME, nitric oxide synthase [NOS] inhibitor) or PD-98059 (inhibitor of mitogen-activated protein kinase kinase 1 and 2 [MEK1/2]). Hypoxia reduced endothelial NOS (eNOS) activity and eNOS phosphorylation at Ser1177, but increased eNOS protein level. Hypoxia increased (1 to 3 hours), but reduced (24 hours) p42/44mapk phosphorylation. Thus, hypoxia-increased extracellular adenosine may result from reduced hENT1-adenosine transport in HUVEC. Hypoxia effect seems not to involve NO, but p42/44mapk may be required for the relatively rapid effect (1 to 3 hours) of hypoxia. These results could be important in diseases where the fetus is exposed to intrauterine environments poor in oxygen, such as intrauterine growth restriction, or where adenosine transport is altered, such as gestational diabetes.

Equilibrative Nucleoside (ENTs) and Cationic Amino Acid (CATs) Transporters: Implications in Foetal Endothelial Dysfunction in Human Pregnancy Diseases

Gestational diabetes (GD, characterized by abnormal D-glucose metabolism), intrauterine growth restriction (IUGR, a disease associated with reduced oxygen delivery (hypoxia) to the foetus), and preeclampsia (PE, a pregnancy complication characterized by high blood pressure, proteinuria and increased vascular resistance), induce foetal endothelial dysfunction with implications in adult life and increase the risk of vascular diseases. Synthesis of nitric oxide (NO) and uptake of L-arginine (the NO synthase (NOS) substrate) and adenosine (a vasoactive endogenous nucleoside) by the umbilical vein endothelium is altered in pregnancies with GD, IUGR or PE. Mechanisms underlying these alterations include differential expression of equilibrative nucleoside transporters (ENTs), cationic amino acid transporters (CATs), and NOS. Modulation of ENTs, CATs, and NOS expression and activity in endothelium involves protein kinase C (PKC), mitogen-activated protein kinases p42 and p44 (p42/44(mapk)), calcium, and phosphatidyl inositol 3 kinase (PI3k), among others. Elevated extracellular D-glucose and hypoxia alter human endothelial function. However, information regarding the transcriptional modulation of ENTs, CATs, and NOS is limited. This review focuses on the effect of transcriptional and post-transcriptional regulatory mechanisms involved in the modulation of ENTs and CATs, and NOS expression and activity, and the consequences for foetal endothelial function in GD, IUGR and PE. The available information will contribute to a better understanding of the cell and molecular basis of the altered vascular endothelial function in these pregnancy diseases and will emphasize the key role of this type of epithelium in placental function and the normal foetal development and growth.

Nitric oxide reduces adenosine transporter ENT1 gene (SLC29A1) promoter activity in human fetal endothelium from gestational diabetes.

Human umbilical vein endothelial cells (HUVEC) from gestational diabetes exhibit reduced adenosine uptake and increased nitric oxide (NO) synthesis. Adenosine transport via human equilibrative nucleoside transporters 1 (hENT1) is reduced by NO by unknown mechanisms in HUVEC. We examined whether gestational diabetes‐reduced adenosine transport results from lower hENT1 gene (SLC29A1) expression. HUVEC from gestational diabetes exhibit reduced SLC29A1 promoter activity when transfected with pGL3‐hENT1−2154 compared with pGL3‐hENT1−1114 constructs, an effect blocked by NG‐nitro‐L‐arginine methyl ester (L‐NAME, NOS inhibitor), but unaltered by S‐nitroso‐N‐acetyl‐L,D‐penicillamine (SNAP, NO donor). In cells from gestational diabetes transfected with pGL3‐hENT1−2154, L‐NAME increased, but SNAP did not alter promoter activity and hENT1 expression. However, in cells from normal pregnancies L‐NAME increased, but SNAP reduced promoter activity and hENT1 expression. Adenovirus‐silenced eNOS expression increased hENT1 expression and activity in cells from normal or gestational diabetic pregnancies. Thus, reduced adenosine transport may result from downregulation of SLC29A1 expression by NO in HUVEC from gestational diabetes. These findings explain the accumulation of extracellular adenosine detected in cultures of HUVEC from gestational diabetes. In addition, fetal endothelial dysfunction could be involved in the abnormal fetal development and growth seen in gestational diabetes. J. Cell. Physiol. 208: 451–460, 2006.

Inhibition of Nitrobenzylthioinosine-Sensitive Adenosine Transport by Elevated d-Glucose Involves Activation of P2Y2 Purinoceptors in Human Umbilical Vein Endothelial Cells

Chronic incubation with elevated d-glucose reduces adenosine transport in endothelial cells. In this study, exposure of human umbilical vein endothelial cells to 25 mmol/L d-glucose or 100 &mgr;mol/L ATP, ATP-&ggr;-S, or UTP, but not ADP or &agr;,&bgr;-methylene ATP, reduced adenosine transport with no change in transport affinity. Inhibition of transport by d-glucose, ATP, and ATP-&ggr;-S was associated with reduced maximal binding, with no changes in the apparent dissociation constant for nitrobenzylthioinosine (NBMPR). A significant reduction (≈60±10%, P <0.05; n=6) in the number of human equilibrative NBMPR-sensitive nucleoside transporters (hENT1s) per cell (1.8±0.1×106 in 5 mmol/L d-glucose) and in hENT1 mRNA levels was observed in cells exposed to d-glucose or ATP-&ggr;-S. Incubation with elevated d-glucose, but not with d-mannitol, increased the ATP release by 3±0.2-fold. The effects of d-glucose and nucleotides on the number and activity of hENT1 and hENT1 mRNA were blocked by reactive blue 2 (nonspecific P2Y purinoceptor antagonist), suramin (G&agr;s protein inhibitor), or hexokinase but not by pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (nonselective P2 purinoceptor antagonist). Our findings demonstrate that inhibition of adenosine transport via hENT1 in endothelial cells cultured in 25 mmol/L d-glucose could be due to stimulation of P2Y2 purinoceptors by ATP, which is released from these cells in response to d-glucose. This could be a mechanism to explain in part the vasodilatation observed in the early stages of diabetes mellitus or in response to d-glucose infusion.

Insulin-stimulated L-arginine transport requires SLC7A1 gene expression and is associated with human umbilical vein relaxation.

Insulin causes endothelium‐derived nitric oxide (NO)‐dependent vascular relaxation, and increases L‐arginine transport via cationic amino acid transporter 1 (hCAT‐1) and endothelial NO synthase (eNOS) expression and activity in human umbilical vein endothelium (HUVEC). We studied insulin effect on SLC7A1 gene (hCAT‐1) expression and hCAT‐transport activity role in insulin‐modulated human fetal vascular reactivity. HUVEC were used for L‐arginine transport and L‐[3H]citrulline formation (NOS activity) assays in absence or presence of N‐ethylmaleimide (NEM) or L‐lysine (L‐arginine transport inhibitors). hCAT‐1 protein abundance was estimated by Western blot, mRNA quantification by real time PCR, and SLC7A1 promoter activity by Luciferase activity (−1,606 and −650 bp promoter fragments from ATG). Specific protein 1 (Sp1), and total or phosphorylated eNOS protein was determined by Western blot. Sp1 activity (at four sites between −177 and −105 bp from ATG) was assayed by chromatin immunoprecipitation (ChIP) and vascular reactivity in umbilical vein rings. Insulin increased hCATs–L‐arginine transport, maximal transport capacity (Vmax/Km), and hCAT‐1 expression. NEM and L‐lysine blocked L‐arginine transport. In addition, it was trans‐stimulated (∼7.8‐fold) by L‐lysine in absence of insulin, but unaltered (∼1.4‐fold) in presence of insulin. Sp1 nuclear protein abundance and binding to DNA, and SLC7A1 promoter activity was increased by insulin. Insulin increased NO synthesis and caused endothelium‐dependent vessel relaxation and reduced U46619‐induced contraction, effects blocked by NEM and L‐lysine, and dependent on extracellular L‐arginine. We suggest that insulin induces human umbilical vein relaxation by increasing HUVEC L‐arginine transport via hCATs (likely hCAT‐1) most likely requiring Sp1‐activated SLC7A1 expression. J. Cell. Physiol. 226: 2916–2924, 2011.