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.


Introduction
Hyperglycaemia and diabetes mellitus are pathological conditions associated with foetal endothelial dysfunction [1][2][3][4] and type 2 diabetes mellitus (T2DM) [5] or cardiovascular disease (CVD) [6,7] in adulthood. CVD in patients with diabetes mellitus is associated with the generation of reactive oxygen species (ROS) [8] caused by chronic hyperglycaemia [3] and insulin resistance [9]. Nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) activity and endothelial nitric oxide (NO) synthase (eNOS) uncoupling leads to vascular ROS generation [10], of which superoxide anion (O 2 •-) reduces NO bioavailability, generating peroxynitrite (ONOO -) and resulting in altered vascular endothelial function [11]. NO is synthesised by eNOS from the cationic amino acid L-arginine, which is taken up from the extracellular space by the human cationic amino acid transporter 1 (hCAT-1), a member of the cationic amino acid transporter (CATs) family [11], in human umbilical vein endothelial cells (HUVECs) [12]. Thus, NO bioavailability depends on eNOS activity and hCAT-1 expression and activity [13], as well as ROS levels [11], in this cell type. Elevated extracellular D-glucose increases hCAT-1-mediated L-arginine transport and NO synthesis (the 'L-arginine/NO pathway') [14,15] as well as intracellular ROS generation [16,17], leading to endothelial dysfunction [2]. Insulin causes human umbilical vein endotheliumdependent dilation and increases hCAT-1 expression caused by elevated SLC7A1 (encoding hCAT-1) transcriptional activity [12,14,18] and NO synthesis [12]. Additionally, insulin reverses the gestational diabetes mellitus (GDM) or high D-glucose-associated stimulation of the L-arginine/NO pathway in HUVECs [11]. We hypothesise that insulin has a beneficial antioxidant capacity that reverses the high D-glucose-associated increase in ROS generation. We studied the effect of high extracellular D-glucose on insulin modulation of the L-arginine/NO and NADPH oxidase/O 2 •pathways in fetoplacental vascular reactivity. The results suggest that D-glucose-increased oxidative stress and vascular dysfunction are attenuated by insulin. Thus, insulin likely acts as an antioxidant under conditions of hyperglycaemia, leading to protection of the fetoplacental endothelium in diseases associated with endothelial dysfunction such as GDM.

Ethics statement
This investigation conforms to the principles outlined in the Declaration of Helsinki and has received approval from the Ethics Committee of the Faculty of Medicine of the Pontificia The maximal velocity (V max ) and apparent Michaelis-Menten constant (K m ) of saturable transport were calculated as described [12,14,18].

Immunofluorescence and confocal laser scanning microscopy
The HUVECs were grown on microscope cover slips (

Total RNA isolation and reverse transcription
Total RNA was isolated using the Chomczynski method as previously described [12]. The RNA quality and integrity were ensured by gel visualisation and spectrophotometric analysis (OD 260/280 ), quantified at 260 nm and precipitated to obtain 4 μg/μL RNA. Aliquots (1 μg) of the total RNA were reverse transcribed into cDNA as described [14,18,19].

Actinomycin D effect on hCAT-1 mRNA
Total RNA and protein were isolated from the HUVECs cultured in PCM containing 5 or 25 mmol/L D-glucose (24 hours) in the absence or presence (8 hours) of 1.5 μmol/L actinomycin D (transcription inhibitor) [20] and/or 1 nmol/L insulin. The hCAT-1 mRNA and 28S rRNA were quantified by real-time RT-PCR.

Luciferase assay
The electroporated cells were lysed in 200 μL of passive lysis buffer (Promega), and firefly and Renilla luciferase activity were measured using the Dual-Luciferase Reporter Assay System (Promega) in a Sirius luminometer (Berthold Detection System; Oak Ridge, TN, USA) [19,20].
The column was then equilibrated with the mobile phase (5% methanol, 95% water) for 40 minutes with a flow rate of 1 mL/minute. Fluorescence was monitored at excitation and emission wavelengths of 350 and 450 nm, respectively, by using the fluorescent detector (FP 2020 Plus, Jasco, Japan) of the HPLC system. Biopterins were quantified against the standard curve 0-100 nmol/L L-biopterin (Sigma-Aldrich). The chromatograms were obtained and analysed by using the software ChromPass 1.7 (ChromPass Chromatography Data System, Jasco, Japan). BH 4 was determined as the difference between the areas under the curve in chromatograms for total biopterins and BH 2 + biopterin. Values for BH 4 level are given in pmol/μg protein.

Statistical analysis
The values are the mean ± S.E.M. for different cell cultures (with 3-4 replicates) from an equal number of placentas (n = 33). The data reported in this study describe a normal standard distribution. The comparisons between two or more groups were performed using Student's unpaired t-test and analysis of variance (ANOVA), respectively. If the ANOVA demonstrated a significant interaction between the variables, post hoc analyses were performed by the multiplecomparison Bonferroni correction test. The GraphPad Instat 3.0b and GraphPad Prism 6.0f (GraphPad Software, Inc., San Diego, CA, USA) statistical software packages were used for the data analysis. P<0.05 was considered significant.

High D-glucose increases L-arginine uptake and hCAT-1 expression
High D-glucose increased L-arginine transport in a concentration-and time-dependent manner ( Fig 1A). The D-glucose effect was biphasic, with an initial increase from 2 hours of   significant increase in transport by 25 mmol/L D-glucose (hereafter referred as 'high D-glucose') was observed starting at approximately 2 hours of incubation and maintained for at least 24 hours, we assayed the insulin effect in the cells incubated with this concentration of D-glucose for 24 hours. Additionally, hCAT-1 mRNA expression and protein abundance increased in a biphasic manner upon high D-glucose treatment (Fig 1B). The pp SE 50 and sp SE 50 values of hCAT-1 mRNA expression were lower than the corresponding values for hCAT-1 protein abundance (Table 1).

Insulin blocks D-glucose-increased hCAT-1 activity and expression
We previously reported that insulin increases the V max of L-arginine transport, without altering the apparent K m , in a concentration-dependent manner in HUVECs cultured in 5 mmol/L Dglucose [12]. The high D-glucose effect was reflected in a higher V max /K m , with a maximal transport increase at 1 nmol/L insulin [12]. Insulin blocked the increase in L-arginine transport caused by high D-glucose in a concentration-dependent manner by decreasing the V max (Fig 2A) and V max /K m (Table 1) values of transport, with a half-maximal inhibitory concentration (IC 50 ) for an insulin effect of 0.11 ± 0.007 nmol/L ( Fig 2B). Insulin restored the increase in hCAT-1 protein abundance (Fig 3A), hCAT-1 mRNA expression ( Fig 3B) and fluorescence (Fig 3C). In addition, high D-glucose and insulin increased these parameters in cells in 5 mmol/L D-glucose. Actinomycin D blocked the effect of high D-glucose on hCAT-1 mRNA expression (Fig 3D). In addition, incubation of cells with high D-glucose increased SLC7A1 promoter transcriptional activity in cells transfected with either pGL3-hCAT1 -650 (1.39 ± 0.08-fold) or pGL3-hCAT1 -1606 (1.46 ± 0.05-fold) constructs compared with non-transfected cells in 5 mmol/L D-glucose (Fig 3E). Insulin increased the transcriptional activity only in cells transfected with pGL3-hCAT1 -1606 (1.37 ± 0.06-fold). However, insulin reverted the effect of high D-glucose on SLC7A1 transcriptional activity for cells containing each construct.
Insulin blocks D-glucose-increased ROS and NO synthesis D-Glucose increased ROS formation (SC 50 = 9.9 ± 0.5 mmol/L), an effect abolished by apocynin and insulin (Fig 4A). High D-glucose also increased O 2 •generation, which was abolished by apocynin, insulin and/or tempol (Fig 4B). High D-glucose and insulin increased the NO level in cells in 5 mmol/L D-glucose; however, insulin but not apocynin or tempol blocked the high D-glucose-mediated increase in the NO level ( Fig 4C).

SOD mimetics block D-glucose-reduced BH 4 level
HUVECs exposed to high D-glucose show lower BH 4 level compared with cells in 5 mmol/L D-glucose, an effect unaltered by insulin or apocynin (Fig 4D). However, the SOD mimetics tempol and Mn(III)TMPyP increased the BH 4 level to comparable values in cells in 5 or 25 mmol/L D-glucose.

Insulin and tempol block D-glucose-increased p42/44 mapk phosphorylation
In cells in 5 mmol/L D-glucose insulin increased p42/44 mapk (Fig 5A) and Akt (Fig 5B) phosphorylation, but tempol did not alter the phosphorylation of these molecules under this experimental condition. High D-glucose increased p42/44 mapk , but not Akt phosphorylation, which was reduced by insulin or tempol.

Role of NADPH oxidase on hCAT-1 expression
Apocynin blocked the increase in hCAT-1 protein abundance (Fig 5C), hCAT-1 mRNA expression (Fig 5D), and SLC7A1 promoter activity caused by high D-glucose in cells transfected with the pGL3-hCAT1 -650 construct ( Fig 5E). However, the increase in these parameters caused by insulin in cells in 5 mmol/L D-glucose was unaltered by this inhibitor.

Effect of D-glucose on vascular reactivity
Incubation of human umbilical vein rings with high D-glucose for 24 hours caused a higher maximal contraction in response to U46619, compared with vein rings in 5 mmol/L D-glucose (Fig 6A). The SC 50 for the U46619 effect in high D-glucose (24.5 ± 0.6 nmol/L) was lower (P < 0.05) than that in 5 mmol/L D-glucose (62.3 ± 0.9 nmol/L). Insulin, apocynin and tempol blocked the U46619-increased contraction in high D-glucose (Fig 6B). Umbilical vein rings in high D-glucose show reduced maximal dilation in response to CGRP compared with vein rings in 5 mmol/L D-glucose (Fig 6C). The half-effective concentration (EC 50 ) for the CGRP vasodilation was similar in both experimental conditions (9.7 ± 0.2 and 9.6 ± 0.2 nmol/L for 5 and 25 mmol/L D-glucose, respectively). Insulin, but not apocynin or tempol increased vein rings dilation in 5 mmol/L D-glucose; however, these molecules reversed the high D-glucose-decreased vein rings dilation (Fig 6D). In addition, 20 mmol/L D-mannitol + 5 mmol/L D-glucose did not alter vascular reactivity to U46619 or CGRP (not shown).

Discussion
This study shows that high extracellular D-glucose increases L-arginine transport, NO synthesis and O 2 •generation through eNOS and NADPH oxidase activation. Additionally, high Dglucose increased the contractile response to U46619 in umbilical vein rings. Insulin reversed these effects of high D-glucose, leading to normal hCAT-1 expression, NO synthesis, O 2 •generation and vasorelaxation. Insulin and tempol restored high D-glucose-increased p42/44 mapk activation. Insulin acts as a protective factor for fetoplacental vascular dysfunction by reducing the oxidative stress caused by high D-glucose. D-Glucose caused a biphasic increase in L-arginine transport with a half-maximal primary peak stimulation of L-arginine transport ( pp SE 50 ) by 10 and 15 mmol/L D-glucose at longer time of incubation (~2-fold) than for 20 and 25 mmol/L D-glucose. Thus, pp SC 50 at higher and lower D-glucose concentrations may result from different mechanism(s). Cells exposed for 24 hours to high D-glucose exhibited a pp SE 50 value similar to that observed upon the increase in hCAT-1 protein abundance, suggesting that high D-glucose-mediated increased L-arginine transport likely occurred due to a higher hCAT-1 level. Because at the half-maximal secondary peak stimulation of transport ( sp SC 50 ) values were similar for all D-glucose concentrations, either a mixture of mechanisms or different mechanisms may account for this phenomenon; however, there was still an increase in hCAT-1 protein abundance in HUVECs. Interestingly, increased hCAT-1 expression does not seem to be involved in the increased L-arginine transport reported in response to 25 mmol/L D-glucose for 48 hours in human aortic endothelial cells (HAECs) [25]. Thus, modulation of L-arginine transport by high D-glucose could be different depending on the source of human macrovascular endothelium. High D-glucose also caused a biphasic increase in hCAT-1 mRNA expression with pp SE 50 and sp SE 50 occurring earlier than for L-arginine transport (72 ± 12 and 78 ± 8 minutes, respectively), and pp SE 50 abundance as in A. Lower panel: P~Akt/Total Akt ratio densitometries normalized to 1 in 5 mmol/L D-glucose in the absence of insulin or tempol. C, Western blot for hCAT-1 protein abundance in the absence (-) or presence of insulin, without (-Apo) (Control) or with (+Apo) 100 μmol/L apocynin. Lower panel: hCAT-1/β-actin ratio densitometries normalized to 1 in 5 mmol/L D-glucose in the absence of insulin or apocynin. Bars are cells without (-Apocynin) or with 100 μmol/L apocynin (+Apocynin). D, Number of copies of hCAT-1 mRNA and 28S rRNA (internal reference) in cells as in C. E, Luciferase reporter construct activity for cells transfected with a truncated SLC7A1 promoter (-650 bp (pGL3-hCAT-1 -650 ) from ATG) as in C. In A occurring earlier than hCAT-1 protein abundance (78 ± 7 minutes). No reports are available that address the potential half-life of the hCAT-1 protein and hCAT-1 mRNA in human endothelial cells [11]. The CAT-1 mRNA half-life was 90-250 minutes in the rat hepatoma FTO2B cell line [26] and approximately 75 minutes in the stably transfected rat glioma C6 cell line [27]. In addition, CAT-1 mRNA turnover in response to amino acid deprivation increased between 6-12 hours. Thus, hCAT-1 mRNA turnover would be approximately 12 hours in response to high D-glucose, with a half-life of 1-2 hours in HUVECs in 5 mmol/L D-glucose in the absence of insulin. After challenge with high D-glucose, the hCAT-1 mRNA turnover kinetics could be extended to approximately 6-12 hour cycles. Insulin increases L-arginine transport and NO synthesis involving protein kinase B (PKB)/ Akt in HAECs [25]. Incubation of HAECs with 25 mmol/L D-glucose increased L-arginine transport, which is blocked by 1 nmol/L insulin. High D-glucose-increased hCAT-1 protein abundance, and activity in HUVECs was also blocked by insulin; however, the insulin effect was observed at a lower concentration (~10-fold) in HUVECs than in HAECs, with IC 50 close to insulinemia in human umbilical vein (~0.041 nmol/L) [28] and whole umbilical cord (~0.025 nmol/L) [29,30]. Thus, HUVECs are highly sensitive to insulin compared with HAECs. Insulin also restored the high D-glucose-mediated increase in V max /K m , suggesting a lower number of membrane transporters rather than reduced affinity of a fixed number of transporters [11,18]. Indeed, insulin restored hCAT-1 protein abundance and the plasma membrane/cytosol distribution. Temporality in changes mediated by high D-glucose in hCAT-1 protein abundance and activity are similar; thus, increased transport caused by high D-glucose could result from higher hCAT-1 protein abundance, preferentially localised at the plasma membrane in HUVECs. High D-glucose-associated changes in hCAT-1 expression could result from transcriptional and/or post-transcriptional modulation by insulin. In fact, the insulin effect requires transcriptional regulatory factors acting at the -650 bp fragment of the promoter region of SLC7A1. Specific protein 1 (Sp1) mediates insulin effects in several cell types, similar to other TATA-less promoters [11,31,32], including increased SLC7A1 transcriptional activity in HUVECs [12]. SLC7A1 exhibits at least four Sp1 consensus sequences between -177 and -105 bp from its translation start point in HUVECs [11,12], making it likely that insulin-restored SLC7A1 promoter activity results from reduced Sp1 activity. However, other transcription factor(s) that reduce SLC7A1 transcriptional activity, such as the C/EBP homologous protein 10 (CHOP) as reported in C6 rat glioma cells [33], may be involved in mediating insulin effects.
NADPH oxidase generates ROS in HUVECs exposed to hyperglycemia [18]. We showed that high D-glucose increases ROS generation with a SC 50 value of approximately 11 mmol/L, which is similar to the stimulatory effect by high D-glucose on L-arginine transport (SC 50 of 13 mmol/L). Thus, the high D-glucose-mediated increase in ROS generation could lead to increased L-arginine transport in HUVECs. Because ROS generation was blocked by apocynin, high D-glucose activation of NAPDH oxidase is feasible, agreeing with findings in human umbilical artery endothelium exposed to 30 mmol/L D-glucose [34] and in EAhy926 cells (immortalised endothelial cell line) exposed to 35 mmol/L D-glucose [35]. Apocynin blocked the high D-glucose-mediated increase in ROS and O 2 •generation in similar proportions (~69 and 83%, respectively), suggesting that O 2 •is the main form of NADPH oxidase-generated ROS (~82%). The latter was further supported by the results showing that the SOD mimetic tempol reduced the effect of high D-glucose in O 2 •generation. Interestingly, insulin blocked ROS and O 2 •generation under high D-glucose; thus, a potential anti-oxidative stress role for insulin by decreasing NADPH oxidase activity is feasible in HUVECs, which agrees with findings in mesenteric arterioles from diabetic rats [36]. However, since HUVECs exposed to high D-glucose increases mitochondrial O 2 •generation [37], the possibility that insulin also modulates this source of O 2 •generation is likely.
Insulin and tempol reversed the high D-glucose-increase in p42/44 mapk phosphorylation, suggesting that cell signalling mediated by activation of these protein kinases could result from a higher O 2 •generation under high D-glucose. In addition, a potential protective effect of insulin in this phenomenon is feasible. On the contrary, since high D-glucose did not alter Akt phosphorylation it is likely that this molecule is not involved in the response to this environmental condition enriched in O 2 •in HUVECs from normal pregnancies. The insulin response in this study is similar to that reported in primary cultures of HUVECs from GDM pregnancies [38]. Since GDM associates with overexpression of insulin receptor A (IR-A) form in HUVECs, and activation of these receptors leads to preferential p42/44 mapk phosphorylation compared with Akt phosphorylation in HUVECs from normal pregnancies, it is feasible that insulin mediates similar mechanisms to restore p42/44 mapk associated cell signalling in a high D-glucose environment and in GDM. High D-glucose caused comparable increases in NO and ROS generation; thus, these phenomena are linked in HUVECs, agreeing with a report on this cell type when it was incubated with 75 mmol/L D-glucose [39]. The high D-glucose-mediated increase in NO synthesis is NADPH oxidase-independent, as apocynin was ineffective. However, because insulin blocked the D-glucose effect on NO synthesis and ROS generation, an alternative mechanism for the response to this hormone in HUVECs is likely. Thus, insulin could be equally active in preventing O 2 •oxidative stress in HUVECs under high D-glucose conditions. eNOS uncoupling due to imbalanced tetrahydrobiopterin (BH 4 ) and dihydrobiopterin (BH 2 ) levels (BH 2 /BH 4 > 1) leads to O 2 •generation [40]. •generation by reducing the eNOS dimer/monomer ratio (i.e., uncoupled eNOS) and NO synthesis in EA.hy926 cells exposed to 35 mmol/L D-glucose [35], (c) restoration of eNOS dimerization and function by BH 4 administration in diabetic rats [36] and (d) restoration by apocynin of the diabetes mellitus-increased eNOS-derived O 2 •in mice [41]. Taking in consideration that incubation of HUVECs with~22.5 mmol/L D-glucose causes an estimated increase of~2.3 fold of the peroxynitrite (ONOO -) level [42], a metabolite that results from the reaction between NO and O 2

•-
, and since in our study the increase in NO level (~1.8 fold) and O 2 •generation (~2.1 fold) were similar, we speculate on the possibility that ONOOgeneration is likely increased in~2 fold in HUVECs exposed to high D-glucose. In addition, insulin caused a comparable reduction (~32%) in NO level and O 2 •generation in cells in high D-glucose, suggesting the possibility that this hormone could also cause a reduction in ONOOgeneration in HUVECs. However, since apocynin or tempol restored the high D-glucose-elevated O 2 •generation, but not the increase in the NO level to values in cells in 5 mmol/L D-glucose, O 2 •generation from NADPH oxidase may not be enough to cause an increase in the ONOOgeneration in high D-glucose in this cell type. Increases in hCAT-1 protein abundance, hCAT-1 mRNA expression and SLC7A1 promoter activity by high D-glucose, but not insulin, were blocked by apocynin, suggesting a NADPH oxidase-dependent mechanism. It has been reported that eNOS activity is associated with higher hCAT-1-mediated L-arginine transport in EA.hy926 cells [13]. Because insulin blocked the increase in V max /K m for L-arginine transport but reduced the high-D-glucose-mediated increase in the NO level by~63%, the high D-glucose effect was partially dependent (~25-30%) on L-arginine transport in HUVECs. In addition, lower hCAT-1 protein abundance compared with reduced V max /K m could be mainly responsible (~75%) for the insulin-mediated reversal of NO synthesis. Our findings also show that insulin reduced hCAT-1 protein abundance at the plasma membrane at high D-glucose concentrations. Because insulin increased hCAT-1 protein abundance in membrane fractions in HUVECs in 5 mmol/L D-glucose [12] and caused a general increase in its cellular distribution in this cell type, reduced hCAT-1 protein abundance at the plasma membrane in cells challenged with high D-glucose could be caused by insulin. These findings support the possibility that not only a reduction in the total protein abundance but also potential hCAT-1 redistribution results from incubation with high D-glucose and insulin. The opposite response to insulin regarding hCAT-1 activity and expression in HUVECs in 5 mmol/L versus 25 mmol/L D-glucose is a finding that is similar to the differential response to this hormone reported for eNOS expression and activity as well as for the human equilibrative nucleoside transporter 1 (hENT1) expression and activity in HUVECs from normal pregnancies compared with GDM pregnancies [38]. The mechanisms behind these opposite effects of insulin involve activation of A 1 and A 2A adenosine receptors leading to modulation of the biological effect of insulin in HUVECs from normal pregnancies [18], GDM pregnancies [38], and in preeclampsia [43]. These mechanisms were not evaluated in this study; however, it is suggested that insulin could use an intracellular metabolic machinery to modulate expression of SLC29A7 and hCAT-1 protein that is different in HUVECs in a normal or high D-glucose environment, as reported in GDM [44].
Umbilical vein contraction was caused by the thromboxane A 2 mimetic U46619, as reported in human chorionic plate arteries [24]. The U46619 maximal contraction in high D-glucose conditions was higher (~1.8-fold) compared with 5 mmol/L D-glucose, suggesting that reduced bioavailability of agents causing vasorelaxation or increased agents causing vasoconstriction may be responsible for this phenomenon. Interestingly, U46619 half-maximal constriction in high D-glucose was higher than in 5 mmol/L D-glucose ( 25 mmol/L SC 50 / 5 mmol/L SC 50 = 2.5), suggesting that under high D-glucose, umbilical vein rings are more reactive to this molecule than preparations in a physiological concentration of D-glucose. It is likely that increased contraction of umbilical vein rings in high D-glucose conditions occurred due to increased ROS and O 2 •generation because this vascular response was blocked by insulin, apocynin and tempol.
Since similar results were found in the response of umbilical vein rings to CGRP (a preferential endothelium-dependent vasodilator), not only constriction, but also dilation are altered involving similar mechanisms in terms of oxidative stress in vein rings exposed to high D-glucose. Interestingly, since CGRP half-maximal dilation in high D-glucose was similar to that in 5 mmol/L D-glucose ( 25 mmol/L EC 50 / 5 mmol/L EC 50 = 1.02), a reduced sensibility to vasodilators is unlikely to be the cause of the high D-glucose-increased vasoconstriction in human umbilical vein rings. HUVECs exposure to high D-glucose increases L-arginine transport (Fig 7), likely resulting from higher hCAT-1 expression and protein abundance in the plasma membrane. This mechanism could be an adaptive response of HUVECs to higher ROS and O 2 •generation from high D-glucose-activated NADPH oxidase. In parallel, high D-glucose increased NO synthesis, which was independent of NADPH oxidase activation. Insulin reversed the high D-glucosemediated alterations in L-arginine transport involving the modulation of SLC7A1 gene expression, leading to altered umbilical vein reactivity. Modulation of hCAT-1 expression and activity is key to maintaining umbilical vein tone and endothelial function. Most diseases of pregnancy progress with oxidative stress leading to altered placental vascular reactivity [11]. Thus, management of potential pro-oxidative stress conditions is necessary to prevent fetoplacental endothelial dysfunction, thus ensuring an adequate supply of nutrients to the growing foetus.