Rocío Salsoso

Insulin Reverses D-Glucose–Increased Nitric Oxide and Reactive Oxygen Species Generation in Human Umbilical Vein Endothelial Cells

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.

Maternal Hypercholesterolemia in Pregnancy Associates With Umbilical Vein Endothelial Dysfunction Role of Endothelial Nitric Oxide Synthase and Arginase II

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 <280 mg/dL was considered as maternal physiological hypercholesterolemia (n=46 women) and ≥280 mg/dL as MSPH (n=28 women). Maternal but not fetal total cholesterol and low-density lipoprotein-cholesterol levels were elevated in MSPH. Umbilical veins were used for vascular reactivity assays (wire myography), and primary cultures of umbilical vein endothelial cells to determine arginase, endothelial NO synthase (eNOS), and human cationic amino acid transporter 1 and human cationic amino acid transporter 2A/B expression and activity. MSPH reduced calcitonine gene–related peptide-umbilical vein relaxation and increased intima/media ratio (histochemistry), as well as reduced eNOS activity (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.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 <280 mg/dL was considered as maternal physiological hypercholesterolemia (n=46 women) and ≥280 mg/dL as MSPH (n=28 women). Maternal but not fetal total cholesterol and low-density lipoprotein-cholesterol levels were elevated in MSPH. Umbilical veins were used for vascular reactivity assays (wire myography), and primary cultures of umbilical vein endothelial cells to determine arginase, endothelial NO synthase (eNOS), and human cationic amino acid transporter 1 and human cationic amino acid transporter 2A/B expression and activity. MSPH reduced calcitonine gene–related peptide-umbilical vein relaxation and increased intima/media ratio (histochemistry), as well as reduced eNOS activity (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. # Significance {#article-title-49}

Insulin requires normal expression and signaling of insulin receptor A to reverse gestational diabetes-reduced adenosine transport in human umbilical vein endothelium

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 (p44/42mapk) 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/ 42mapk:Akt activity (2.7‐fold) ratios. Insulin reversed GDM‐reduced hENT1 expression and maximal transport capacity(Vmax/Km),andGDM‐increased IR‐A/IR‐B mRNA expression and p44/42mapk:Akt activity ratios to values in normal pregnancies. Insulins effect was abolished in IR‐A or IR‐A/B knockdown cells. Thus, insulin requires normal IR‐A expression and p44/42mapk/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.‐Westermeier, F., Salomon, C., Farías, M., Arroyo, P., Fuenzalida, B., Sáez, T., Salsoso, R., Sanhueza, C., Guzmán‐Gutiérrez, E., Pardo, F., Leiva, A., Sobrevia, L. Insulin requires normal expression and signaling of insulin receptor A to reverse gestational diabetes‐reduced adenosine transport in human umbilical vein endothelium. FASEB J. 29, 37–49 (2015). www.fasebj.org

Insulin therapy and fetoplacental vascular function in gestational diabetes mellitus

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.

Role of Insulin and Adenosine in the Human Placenta Microvascular and Macrovascular Endothelial Cell Dysfunction in Gestational Diabetes Mellitus

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.

Insulin restores l-arginine transport requiring adenosine receptors activation in umbilical vein endothelium from late-onset preeclampsia

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 Ser¹¹⁷⁷- or Thr⁴⁹⁵-phosphorylated eNOS were detected via Western blot, and L-arginine transport (0-1000 μmol/L L-arginine, 3 μCi/mL L-[³H]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.

Nitric Oxide is a Central Common Metabolite in Vascular Dysfunction Associated with Diseases of Human Pregnancy

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 supraphysiological gestational weight gain and fetoplacental vascular dysfunction

Objective: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.Methods: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 gain >16 kg, rates of weight gain >0.42 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 Ser1177- or Thr495-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 [3H]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.Results:spGWG associates with reduced NOS activity-dependent dilation of vein rings (P=0.001), lower eNOS expression and higher Thr495 (P=0.044), but unaltered Ser1177eNOS 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.Conclusion: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.

Insulin/adenosine axis linked signalling

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 (A1AR, A2AAR, A2BAR, A3AR), of which A2AAR and A2BAR 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 A1AR 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 A2AAR and A2BAR play a major role. Other studies show that A1AR activation could reduce insulin release from pancreatic β-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.

Adenosine and preeclampsia

Adenosine is an endogenous nucleoside with pleiotropic effects in different physiological processes including circulation, renal blood flow, immune function, or glucose homeostasis. Changes in adenosine membrane transporters, adenosine receptors, and corresponding intracellular signalling network associate with development of pathologies of pregnancy, including preeclampsia. Preeclampsia is a cause of maternal and perinatal morbidity and mortality affecting 3-5% of pregnancies. Since the proposed mechanisms of preeclampsia development include adenosine-dependent biological effects, adenosine membrane transporters and receptors, and the associated signalling mechanisms might play a role in the pathophysiology of preeclampsia. Preeclampsia associates with increased adenosine concentration in the maternal blood and placental tissue, likely due to local hypoxia and ischemia (although not directly demonstrated), microthrombosis, increased catecholamine release, and platelet activation. In addition, abnormal expression and function of equilibrative nucleoside transporters is described in foetoplacental tissues from preeclampsia; however, the role of adenosine receptors in the aetiology of this disease is not well understood. Adenosine receptors activation may be related to abnormal trophoblast invasion, angiogenesis, and ischemia/reperfusion mechanisms in the placenta from preeclampsia. These mechanisms may explain only a low fraction of the associated abnormal transformation of spiral arteries in preeclampsia, triggering cellular stress and inflammatory mediators release from the placenta to the maternal circulation. Although increased adenosine concentration in preeclampsia may be a compensatory or adaptive mechanism favouring placental angiogenesis, a poor angiogenic state is found in preeclampsia. Thus, preeclampsia-associated complications might affect the cell response to adenosine due to altered expression and activity of adenosine receptors, membrane transporters, or cell signalling mechanisms. This review summarizes the evidence available on the potential involvement of the adenosine in the clinical, pathophysiology, and therapeutic features of preeclampsia.