Current Vascular Pharmacology, 2009, 7, 435-449 435

1570-1611/09 $55.00+.00 © 2009 Bentham Science Publishers Ltd.

Equilibrative Nucleoside Transporters in Fetal Endothelial Dysfunction in Diabetes Mellitus and Hyperglycaemia

Francisco Westermeier1,†

, Carlos Puebla1,†

, José Luis Vega1, Marcelo Farías

1, Carlos Escudero

1,2,

Paola Casanello1 and Luis Sobrevia

1,*

1Cellular and Molecular Physiology Laboratory (CMPL) and Perinatology Research Laboratory (PRL), Department of

Obstetrics and Gynaecology, Medical Research Centre (CIM), School of Medicine, Faculty of Medicine, Pontificia

Universidad Católica de Chile, P.O. Box 114-D, Santiago, Chile; 2Department of Basic Sciences, Faculty of Sciences,

Universidad del Bio-Bio, Chillán, Chile

Abstract: Diabetes mellitus types 1 and 2, and gestational diabetes are characterized by abnormal D-glucose metabolism

and hyperglycaemia, and induce foetal endothelial dysfunction with implications in adult life increasing the risk of vascu-

lar diseases. Synthesis of nitric oxide (NO) and uptake of L-arginine (i.e. the L-arginine/NO signalling pathway) and

adenosine (a vasoactive endogenous nucleoside) by the umbilical vein endothelium is altered in pathological pregnancies,

including pregnancies with pre-established diabetes mellitus or in gestational diabetes. The mechanisms underlying these

alterations include differential expression of equilibrative nucleoside transporters (ENTs), amino acid transporters and NO

synthases (NOS). Modulation of ENTs and NOS expression and activity in endothelium involves several signalling mole-

cules, including protein kinase C, mitogen-activated protein kinases p42 and p44, calcium and phosphatidyl inositol 3

kinase. Elevated extracellular D-glucose and diabetes alters human endothelial function. However, information regarding

modulation the transport capacity as well as expression of ENTs is limited. This review focuses on the effect of diabetes

mellitus and gestational diabetes, and hyperglycaemia on the reported mechanisms described for transcriptional and post-

transcriptional regulation of ENTs, and the potential consequences for foetal endothelial function in these pathologies.

Recent available information regarding functional consequences of an abnormal environment on the functionality of the

endothelium from microvasculature of the human placenta is mentioned. The available information is scarce, but it could

contribute to a better understanding of the cell and molecular basis of the altered vascular endothelial function in this

pathological conditions, emphasizing the key role of this type of epithelium in fetal-placental function and the normal foe-

tal development and growth.

Keywords: Diabetes, gestational diabetes, nucleoside, transport, human, endothelium.

INTRODUCTION

Endothelial function is characterized by the capacity of endothelial cells to synthesize and release vasodilators (in-cluding adenosine or nitric oxide (NO)) and vasoconstrictors (including angiotensin or acetylcholine). Pregestational dia-betes mellitus (type 1 or 2) and gestational diabetes are pa-thologies characterized by endothelial dysfunction which have been associated to altered synthesis of NO and uptake of the cationic amino acid L-arginine (i.e. L-arginine/NO signalling pathway) [1-7]. Even when most studies are not conclusive regarding the mechanisms behind these effects of diabetes, it is accepted that it is the result of altered multiple metabolic mechanisms which includes sensitivity of endo-thelium to vasoactive molecules such as adenosine or insulin in the human fetal endothelium [5-8] and human cardiomio-cytes [9].

Deleterious effect of diabetes mellitus in pregnancy re-sults in foetal vascular dysfunction leading to altered blood

*Address correspondence to this author at the Cellular and Molecular

Physiology Laboratory (CMPL), Department of Obstetrics and Gynaecol-ogy, Medical Research Centre (CIM), School of Medicine, Faculty of Medi-

cine, Pontificia Universidad Católica de Chile, P.O. Box 114-D, Santiago, Chile; Tel: 562-3548116; Fax: 562-6321924; E-mail: sobrevia@med.puc.cl † These authors contributed equally.

flow in the placenta-umbilical cord unit [2, 3, 5-7, 10, 11]. Increased L-arginine transport (the substrate for NO synthe-sis) and expression and activity of endothelial NO synthase (NOS) has been reported in human umbilical vein endothe-lial cells (HUVEC) from gestational diabetes, a phenomenon where adenosine seems to play a determinant role triggering these alterations in this type of endothelium from the mac-rovasculature. This potential relationship between adenosine, acting through membrane receptors, L-arginine transport and NO synthesis was described as ALANO (Adenosine, L-Arginine, Nitric Oxide) pathway for this cell type in this disease [5]. Interestingly, there is no information regarding the mechanisms by which human placenta circulation at the microvascular bed is modulated in terms of adenosine and other vasoactive molecules in diabetes mellitus or other pregnancy diseases, such as pre-eclampsia or intrauterine growth restriction (IUGR) [6, 7]. Microvascular endothelial cells from human placenta (hPMEC) express L-arginine transporters, which apparently drive NO synthesis via the inducible NOS isoform (iNOS) [12], scarce literature is available regarding this phenomenon in this cell type [6, 12]. In addition, nothing is known regarding these mechanisms in pathologies of the pregnancy, including diabetes mellitus. Interestingly, even when some studies have reported altered plasma levels of adenosine in the diabetic mother and in the

436 Current Vascular Pharmacology, 2009, Vol. 7, No. 4 Westermeier et al.

umbilical vein blood, information regarding cellular and mo-lecular mechanisms responsible of these changes in human endothelium is limited [3, 5-7, 13]. There are few reports in the literature showing that endothelial cells exhibit altera-tions in their capacity to take up or release adenosine via the Na

+-independent equilibrative nucleoside transporters

(hENTs) in response to several stimuli, including elevated D-glucose [14-16], reduced oxygen (O2) availability (i.e. hy-poxia) [17, 18], insulin [19], TGF-ß1 [20] or pathologies such as gestational diabetes [2, 5, 6, 8, 21], intrauterine growth restriction [6, 18] and preeclampsia [7, 12]. Thus, since adenosine is a key modulator of the L-arginine/NO signalling pathway in HUVEC [2, 5-7] and several other cell types [5, 22, 23], this review will describe the effect of dia-betes mellitus type 1 and type 2, and gestational diabetes, as well as elevated extracellular D-glucose concentrations on expression and activity of hENTs in human endothelial cells from the feto-placental unit.

EQUILIBRATIVE NUCLEOSIDE TRANSPORTERS

(ENTS)

Adenosine removal from extracellular space is mediated by nucleoside transporters, which have been grouped in two families: the Equilibrative Nucleoside Transporters (ENTs) and the Concentrative Nucleoside Transporters (CNTs) [5-7, 22, 24]. At present, CNTs expression and Na

+-dependent

transport activity, a characteristic of these types of mem-brane transporters, have not been described in endothelial cells [6, 7, 22, 25, 26]. However, a series of studies have characterized adenosine transport via ENTs in this cell type [2, 5-7]. Four members of the ENTs family of solute carriers (SLC29A genes) have been cloned from human tissues, i.e. hENT1, hENT2, hENT3 and hENT4, all of which transport adenosine and exhibit a likely 11-transmembrane helix to-pology [6, 22, 23]. In primary cultures of HUVEC it has been reported that adenosine transport is mainly (~80%) me-diated by the human ENT1 system (hENT1) [8, 21, 27]. The remaining adenosine transport (~20%) seems to be mediated by hENT2 [18, 21]. Recent reports show that these proteins are also expressed in hPMEC, however contribution of hENT1 and hENT2 to total adenosine transport in this cell type is similar [7]. These results are complemented by find-ings showing that both hENT1 and hENT2 mRNA and pro-teins have been detected in these two different cell types [6, 7, 18, 27, 28]. The contribution of hENT3 and hENT4 to nucleoside uptake in endothelial cells is uncertain [6, 7, 29]. ENT3 has been recognised as a lysosomal transporter that will not contribute significantly to modulate the extracellular concentration of nucleosides. ENT4, a relatively new mem-ber of the ENTs, has been described in HUVEC [30], but this nucleoside transporter seems to function at an optimal pH ~5.5, making unlikely that these membrane transporters will contribute to the overall uptake of adenosine by endothelial cells under a normal plasma physiological pH.

The nucleoside transporters isoform hENT1 is a protein of 456 amino acids, encoded by SLC29A1 located at the lo-cus 6p21.2-p21.1. These transporters exhibit apparent Km values in the range of 50-200 M and transport purine and pyrimidine nucleosides, including uptake of the antiviral and anticancer nucleosides cytarabine, gemcitabine and zalcit-abine. hENT2 is a protein of 456 amino acids and is encoded

by SLC29A2 (locus 11q13). In addition to purine and pyrimidine nucleosides, hENT2 also transport nucleobases, being hypoxanthine one of the most studied, and antiviral and anticancer nucleosides, including zidovudine. hENT2 activity exhibits apparent Km values (40-150 M for adeno-sine) in the same range to those reported for hENT1 medi-ated transport. However, it has been possible to inhibit hENT1-mediated transport with the use of concentrations similar or lower than 1 μM of nitrobenzylthioinosine (NBTI). In addition, higher concentrations of this thiol (>1 μM) inhibit also the hENT2-mediated transport [27, 31]. This pharmacological approach has been determinant in pri-mary cultures of HUVEC where it has been reported that NBTI inhibits adenosine transport via hENT1 with a high selectivity (IC50 ~0.1 nM) compared with hENT2 (IC50 ~1.7

M) [6-8, 27]. A similar result has been found in hPMEC primary cultures [7, 12]. Alternatively, another approach used to differentiate hENT1 from hENT2 mediated transport had been the use of hypoxanthine as an inhibitor of adeno-sine transport. This nucleobase inhibits in ~20% overall adenosine transport (i.e. adenosine transport mediated by hENT1 and hENT2 simultaneously) in HUVEC and ~45% overall adenosine transport hPMEC, and abolishes the re-maining adenosine transport in cells exposed to 1 μM NBTI. Thus, taking apart hENT1- and hENT2-mediated transport from overall adenosine transport has been essential to char-acterize the kinetic parameters of these membrane transport-ers when co-expressed in mammalian cells [22, 24].

DIABETES MELLITUS

Diabetes mellitus is a common metabolic disease world-wide affecting approximately 150 million people in year 2000, which is predicted to rise to 220 million in year 2010 [32]. Diabetes mellitus is characterized by chronic hypergly-caemia with disturbances of carbohydrate, fat and protein metabolism resulting from defects in insulin secretion, insu-lin action or both [32]. Diabetes mellitus and the associated complications have become a public health problem. Cardio-vascular disease causes most of the excess morbidity and mortality in diabetes mellitus. Adults with diabetes are at a 2 to 4-fold increased risk of cardiovascular events relative to those without diabetes [33]. It is now recognized that loss of the modulatory role of the endothelium on vascular reactivity may be a critical and initiating factor in the development of diabetic vascular disease [34].

Endothelial dysfunction has received increasing attention as a potential contributor to the pathogenesis of diabetic mi-cro- and macro-angiopathy in diabetes mellitus. Under physiological conditions, there is a balanced release of endo-thelial-derived relaxing and contracting factors, but this deli-cate balance is altered in diabetes, thereby contributing to further progression of vascular damage [35]. Endothelial dysfunction in diabetes originates from as hyperglycaemia and its immediate biochemical sequelae directly alter endo-thelial function increased polyol pathway flux, increased advanced glycation end-product (AGE) formation, activation of protein kinase C (PKC) isoforms and increased hexosa-mine pathway flux [36-39]. Moreover, high D-glucose influ-ences endothelial cell functioning indirectly by the synthesis of growth factors and vasoactive agents in other cells [40].

Endothelial Nucleoside Transporters in Diabetes Current Vascular Pharmacology, 2009, Vol. 7, No. 4 437

Diabetes Mellitus Type 1

Type 1 diabetes mellitus is due primarily to autoimmune-mediated destruction of pancreatic beta cell islets, resulting in absolute insulin deficiency. People with type 1 diabetes mellitus must take exogenous insulin for survival to prevent the development of ketoacidosis, coma and death [32]. Stud-ies

measuring the expression of diabetes related auto-

antibodies in young children from birth suggest that the ap-

pearance of these markers is a major risk for the future de-

velopment of type 1 diabetes [41]. However, the role of auto-

antibodies in the actual pathogenesis of type 1 diabetes has

not been established in humans. In fact, a recent case report

showed the development of type 1 diabetes in a patient with

X linked agammaglobulinaemia, suggesting that autoantibod-

ies are not needed for either the initiation or the progression

of type 1 diabetes [42]. In general, type 1 diabetes is consid-

ered primarily a T cell mediated disease, examination of islet tissue obtained from pancreatic biopsy

from patients with

recent onset type 1 diabetes confirms insulitis, with the pres-

ence of an infiltrate composed of CD4 and CD8 T lympho-

cytes, B lymphocytes, and macrophages, suggesting that

these cells have a role in destruction of the pancreatic -cells [43].

Studies performed in isolated human blood vessels [44] show the existence of endothelial cell dysfunction or reduced response to endothelial NO in diabetes [45]. These results have been confirmed by in vivo studies of diabetic human forearm blood flow [46]. Recent observations using cultured endothelial cells and high D-glucose concentrations show increased apoptosis [47], suggesting the potential endothelial damage by hyperglycemia in diabetic patients. Diabetic mi-crovascular disease, particularly which affecting the eye and kidney, contributes importantly to morbidity [48]. Diabetic microangiopathy is determinant in understanding the basis of the late complications of diabetes. Retinopathy, nephropathy, and diabetic cardiopathy [49] undeniably involve microves-sels. In two genetic models type 1 diabetes, a similar im-paired relaxation has been documented in the aorta [50] and mesenteric arteries [51] of diabetes-prone BB/W or BB/E rats. Similar results are reported for aorta [52, 53] and mes-enteric arteries [54] of the diabetes-prone WBN/Kob rat. Evidence associating reactive oxygen species (ROS) with the etiology of diabetic complications in the vasculature is founded in published studies showing increased generation of ROS and/or decreased endogenous antioxidant defense mechanisms in diabetic endothelial dysfunction. ROS may impair endothelial function through inactivation of NO or by serving as an endothelium-derived contracting factor [55].

Diabetes Mellitus Type 2

In type 2 diabetes, both insulin secretion and action are

impaired, and chronic hyperglycemia is a characteristic fea-

ture of this common metabolic disorder [56]. Unlike type 1 diabetes,

where the disease onset is relatively acute, the

course of type 2 diabetes is slow and the metabolic abnor-

malities that lead to hyperglycemia are established long be-

fore overt diabetes develops [32, 57]. The nature of this pro-

gression must reflect changes in the underlying pathogenetic mechanisms. It is well recognized that type 2 diabetes is the result of two major defects: insulin resistance and defective

-cell function [58]. These two defects, however, contribute to a different extent to the development of the disease. Insu-lin resistance is likely to be almost maximal already in the early stages of the disease. During this phase of the disease glucose tolerance is maintained at the expense of increased insulin secretion, so that insulin-resistant individuals are characterized by compensatory hyperinsulinemia [59, 60]. As soon as compensatory insulin hypersecretion declines impaired glucose tolerance and overt diabetes develop. Hy-perglycaemia and poor metabolic control may then worsen insulin sensitivity, but it is the progressive decline of -cell function that set the pace for increased treatment demand and continuous increase in plasma glucose levels [61].

The role of endothelial dysfunction in type 2 diabetes is more complicated than that for type 1. The effects of aging, hyperlipidemia, hypertension, and other factors add to the complexity of the problem, however, a major pathophysi-ological alteration of type 2 diabetes is insulin resistance [62]. The relationship between insulin resistance and abnor-mal vascular reactivity has been demonstrated in a variety of clinical conditions, and there is evidence that a reduction in insulin resistance is accompanied by improved endothelial function and vice versa [63]. Although the molecular mecha-nisms responsible for the metabolic and vascular abnormali-ties associated with insulin resistance have yet to be entirely elucidated, deficient NO production appears to play an im-portant role. Numerous studies have demonstrated that con-ditions of insulin resistance are commonly associated with endothelial dysfunction and that the exposure of vascular endothelium to high circulating levels of lipids and D-glucose is accompanied by reduced NO availability [64]. These observations have given rise to the theory that endo-thelial dysfunction is both a cause and a consequence of the metabolic disturbances observed in states of insulin resis-tance [65].

Gestational Diabetes

Gestational diabetes is a syndrome characterized by glu-cose intolerance, leading to maternal hyperglycaemia, first recognized during pregnancy, that has been associated with abnormal foetal development and perinatal complications, such as macrosomia, neonatal hypoglycaemia, and neuro-logical disorders [6, 10, 34, 66-68]. Gestational diabetes is characterized by altered D-glucose [66, 69] and adenosine [6, 8, 67, 70] metabolism, and abnormal regulation of the vascular tone in placental and foetal tissues [3, 6, 66, 69, 71-75]. It has been proposed that patients with diabetes mellitus exhibit reduced endothelium dependent vasodilatation due to diminished NO synthesis by the endothelium or lower NO availability possibly due to inactivation by reactive oxygen species (ROS) such as superoxide anion or hydroxyl radicals [66]. However, increased NO synthesis has also been re-ported in human placental veins and arteries [76, 77], and in cultures of HUVEC [8, 21, 78, 79] isolated from gestational diabetic pregnancies. Thus, vascular dysfunction resulting from this pathology may be a consequence of a functional dissociation between the synthesis of NO and its bio-availability to the vascular endothelium and smooth muscle.

Since the initial studies regarding adenosine transport in HUVEC isolated from normal or gestational diabetic preg-

438 Current Vascular Pharmacology, 2009, Vol. 7, No. 4 Westermeier et al.

nancies [80], there is concluding evidence showing that this syndrome is also associated with reduced expression and activity of nucleoside membrane transporter. This phenome-non has been proposed to lead to increased extracellular lev-els of adenosine, which will alter endothelial function and may explain the increased NO synthesis exhibited by HUVEC from this pathology [5, 8]. Thus, it is likely that nucleoside transporters play a crucial role in the modulation of vascular tone in human fetal tissues.

NUCLEOSIDE TRANSPORT IN DIABETES MELLI-TUS

Diabetes Mellitus Types 1 and 2

There is no information on the effect of diabetes mellitus types 1 or 2 on nucleoside transport and/or transporters ex-pression in humans [6] (Table 1). Only few studies in animal models describe that in streptozotocin (STZ)-induced dia-betic rats exhibit reduced expression of rCNTs in almost all nephron segments, an effect that was not prevented by insu-lin treatment which in fact normalized all blood and urine parameters. Another study shows that mRNA levels

of rENT

and rCNT were altered in different tissues of STZ-diabetic rats [81]. In addition, decreased nucleoside uptake was re-ported in hippocampal slices from diabetic rats [82, 83] and reduced ability to release adenosine by diabetic rat cardiac fibroblasts [84]. However, it is clear that the limited informa-tion available addressing adenosine transport in diabetes mellitus type 1 and its potential effect in diabetes-associated endothelial dysfunction has not been accurately studied.

Gestational Diabetes

HUVEC from gestational diabetes exhibit reduced adenosine transport [8, 80], but increased L-arginine trans-port and NO synthesis [8]. Interestingly, incubation of HUVEC from normal pregnancies with 0.1 M NBTI in-duced a reduction in adenosine transport of a similar extent than that exhibited by HUVEC from gestational diabetes in absence of this inhibitor. These results suggest that this pregnancy disease is associated with reduced adenosine up-take due to lower transport activity of hENT1, but not hENT2 in this cell type [27, 28]. Reduced adenosine trans-port has been reported to be associated with lower hENT1 transport capacity (Vmax/Km)[85] in gestational diabetes [8, 80], a result most likely due to down-regulation of hENT1 activity rather than altered intrinsic properties of these trans-porters [5, 6] (Table 1).

Alternatively to a reduced activity of hENT1, a reduced number of NBTI binding sites per endothelial cell (~50%) have been estimated in HUVEC from gestational diabetes [8, 80] or in HUVEC from normal pregnancies exposed to 25 mM D-glucose (~45%) [86, 87] compared with cells from normal pregnancies. In addition, since adenosine uptake effi-ciency (i.e. adenosine molecules per transporter per cell per second) is reported to be unaltered in HUVEC from gesta-tional diabetic pregnancies [5, 8, 80] (Table 2), a reduced hENT1 expression, among its activity, is also likely in this cell type. Similar results have been described in HUVEC isolated from normal pregnancies and exposed for 24 hours to 25 mM D-glucose compared with cells in 5 mM D-glucose as control [14, 87], suggesting that similar mecha-

nisms could be involved in the response to elevated D-glucose and gestational diabetes. In addition, and supporting the latter, HUVEC from gestational diabetes exposed to 25 mM D-glucose did not show further changes to those de-tected in this cell type when cultured in 5 mM D-glucose in terms of maximal number of sites for adenosine at the plasma membrane and efficiency of transport (Table 2).

Recently, it has been possible to characterize for the first time adenosine transport mechanisms in hPMEC primary cultures from normal pregnancies and preeclampsia [7, 12]. This study shows that hPMEC express functional hENT1 and hENT2 and that preeclampsia increases total (i.e. hENT1 + hENT2) and saturable hENT2-mediated adenosine trans-port, but reduces hENT1-mediated transport in this cell type. Alterations in transport were paralleled by increased expres-sion of hENT2, but reduced expression of hENT1. Preeclampsia was also associated with an elevated extracel-lular concentration of adenosine compared with cells from normal pregnancies. In addition the results suggested that A2B adenosine receptors could play a repressor effect on adenosine transport via hENT1, but could induce hENT2-mediated transport in hPMEC from preeclampsia. These mechanisms could become crucial to maintain the physio-logical extracellular adenosine concentration in the mi-crovascular circulation of the human placenta.

A particular analysis of the kinetic parameters and inter-pretation has been proposed in this study, an interpretation that could be used for interpretation of data on nucleoside transport in other cell models or for different pathologies. In fact, a similar analysis has been possible to establish to esti-mate the relative contribution of hENT1 and hENT2 in ges-tational diabetes [21] and in cells exposed to elevated ex-tracellular D-glucose concentration [27] or hypoxia [18]. In brief, it was found that pre-eclampsia was associated with reduced maximal velocity (Vmax) for hENT1-mediated adenosine transport, but increased Vmax for hENT2-mediated transport. This study also shows that apparent Km values were unaltered in preeclampsia, the relative effect of this pathological condition on the maximal transport capacity (Vmax/Km) [3, 88] mediated by hENT2 compared with cells from normal pregnancies (i.e. 1/

N/PE-hENT2F)(see below) was

increased by 2-fold, but for hENT1 (1/N/PE-hENT1

F) it was reduced by 75%. In addition, the relative contribution of hENT1 compared with hENT2 (

hENT1/2F) to the total adeno-

sine transport in cells from normal pregnancies was higher ( 9-fold) than the contribution of this transport system vs. hENT2 estimated in cells from preeclampsia. Relations be-tween maximal transport capacity for ENTs mediated trans-port was used to described this relative contributions. In brief, the relative contribution of hENT1 and hENT2 (

hENT1/2F) [3, 88] to the total adenosine transport (i.e. hENT1

+ hENT2 mediated) was defined as:

hENT1/ 2F =

hENT1Vmax •hENT 2Km

hENT1Km •hENT 2Vmax

where hENT1

Vmax and hENT1

Km are kinetic parameters for satur-able adenosine transport via hENT1, and

hENT2Vmax and

hENT2Km for adenosine transport via hENT2. In addition, the

relative effect of preeclampsia (PE) compared with normal (N) pregnancies on the activity of adenosine transport via

Endothelial Nucleoside Transporters in Diabetes Current Vascular Pharmacology, 2009, Vol. 7, No. 4 439

Table 1. Effect of Diabetes Mellitus on Nucleoside Transporters

Nucleoside

Transporter

Type of

Diabetes

Tissue Transport

Activity

mRNA Level Protein

Abundance

References

STZ-DM Rat kidney – Reduced – [166]

STZ-DM Rat kidney – Unaltered – [81]

STZ-DM Rat fibroblasts – Increased – [84]

STZ-DM Rat liver – Increased – [81]

CNT1

STZ-DM Rat heart – Increased – [81]

STZ-DM Rat kidney – Reduced – [166]

STZ-DM Rat kidney – Unaltered – [81]

STZ-DM Rat fibroblasts – Increased – [84]

STZ-DM Rat B lympho-

cytes

– Increased – [167]

STZ-DM Rat liver – Unaltered – [81]

CNT2

STZ-DM Rat heart – Increased – [81]

CNT3 STZ-DM Rat kidney – Reduced – [166]

GD HUVEC Reduced Reduced Reduced [21, 80, 168]

GD hPMEC – Reduced Reduced F. Westermeier, C. Escudero,

L. Sobrevia (unpublished)

GD HUASMC Increased – – [169]

STZ-DM Rat B lympho-

cytes

– Reduced – [167]

STZ-DM Rat fibroblasts – Reduced – [84]

STZ-DM Rat heart – Reduced – [81]

STZ-DM Rat liver – Reduced – [81]

ENT1

STZ-DM Rat kidney – Unaltered – [81]

GD HUVEC Unaltered – – [21]

STZ-DM Rat B lympho-

cytes

– Reduced – [167]

STZ-DM Rat fibroblasts – Unaltered – [84]

STZ-DM Rat heart – Reduced – [81]

STZ-DM Rat liver – Reduced – [81]

ENT2

STZ-DM Rat kidney – Reduced – [81]

Streptozotocin-induced diabetes mellitus (STZ-DM) or gestational diabetes (GD) on transport activity, protein abundance and mRNA level or number of copies of mRNA for concen-

trative (CNTs) and equilibrative (ENTs) nucleoside transporters. HUVEC: human umbilical vein endothelial cells (primary cultures). hPMEC: human placenta microvascular endo-

thelial cells (primary cultures). HUASMC: human umbilical artery smooth muscle cells, –: not reported.

hENT1 (1/N/PE-hENT1

F) or hENT2 (1/N/PE-hENT2

F) was esti-mated from the maximal transport capacity (Vmax/Km) values for adenosine transport by:

1N / PE hENT1F

=

N hENT1Km •PE hENT1Vmax

N hENT1Vmax •PE hENT1Km

or

1N / PE hENT 2F

=

N hENT 2Km •PE hENT 2Vmax

N hENT 2Vmax •PE hENT 2Km

where N-hENT1

Vmax , N-hENT2

Vmax , N-hENT1

Km and N-hENT2

Km are kinetic parameters for adenosine uptake via hENT1 and hENT2, respectively, in cells from normal pregnancies, and PE-hENT1

Vmax , PE-hENT2

Vmax , PE-hENT1

Km and PE-hENT2

Km for

440 Current Vascular Pharmacology, 2009, Vol. 7, No. 4 Westermeier et al.

adenosine uptake via hENT1 and hENT2, respectively, in cells from preeclampsia [7, 12]. Thus, a similar approach has been used to estimate the relative contribution of a given disease, for example, IUGR (Casanello et al., unpublished data) or gestational diabetes [89] to the membrane transport system capacity for adenosine in HUVEC and hPMEC.

Reduced adenosine transport due to reduced expression and/or activity of ENTs is a phenomenon that could have several physiological implications since a reduced activity could partially explain the abnormally elevated extracellular adenosine concentration detected in culture medium of HUVEC from gestational diabetic (~2 M) compared with normal (~50-500 nM) pregnancies [90, 8, 18], or with the reported adenosine concentration in human umbilical venous blood from normal pregnancies (~500 nM) [91, 92]. This is a phenomenon that seems not to be limited to gestational dia-betes, but also identified in preeclampsia; however, there are not reports regarding molecular mechanisms addressing the effect of gestational diabetes on adenosine catabolism in the feto-placental tissue [93-95]. Unfortunately, as far as we know there is not information regarding plasma levels of adenosine in gestational diabetic patients and human fetal blood [6, 7, 29].

Gestational diabetes is also associated with reduced hENT1 protein level compared with normal cells [21], sug-gesting that reduced hENT1-adenosine transport could be related to reduced hENT1 protein abundance in this cell type, a phenomenon not due to hENT1 protein degradation [21]. This phenomenon support the possibility that reduced maximal velocity of adenosine transport in HUVEC from gestational diabetes could be in fact due to reduced expres-sion of these proteins in the plasma membrane (Table 2). In fact, recent information suggests that hENT1-EGFP fusion protein has been relocated to intracellular compartments in

HUVEC from gestational diabetes; however, the mecha-nisms are unknown [89].

Recent reports show that a region of the promoter of SLC29A1 gene has been cloned in primary cultures of HUVEC [21] and in the human cell line microvascular endo-thelial cells 1 (HMEC-1) [96]. These studies have opened the possibility of understanding pre-transcriptional mechanisms associated with modulation of adenosine transporters in en-dothelial cells. A sequence spanning from -756 bp to the major transcription start site of ENT1 promoter was ampli-fied in HMEC-1, where a sequence flanking -653 to -721 bp seems to be reactive to hypoxia inhibiting expression of SLC29A1. A wider sequence spanning from -3198 bp to ATG translation start codon of SLC29A1, using genomic DNA from primary cultures of HUVEC, as template was also characterized [28]. In this study it was proposed that the region spanning from -1114 to -795 bp of SLC29A1 pro-moter confers gestational diabetes-associated stimulatory activity. In addition, the region spanning from -2154 to -1114 bp of SLC29A1 promoter may lack of these DNA con-sensus motifs for stimulatory transcription factor(s) or, alter-natively, may contain sequences for inhibitory transcription factor(s) leading to down-regulation of SLC29A1 promoter activity in HUVEC from gestational diabetic pregnancies. Interestingly, NO induces a reduced SLC29A1 transcription due to repression at the promoter region spanning -2154 to -1114 bp in HUVEC from gestational diabetes. The mecha-nism beyond these potential effects of NO is unclear, but relevant evidence has been communicated regarding the in-volvement of hCHOP-C/EBP transcription factor [89], a protein that is activated by NO in other cell types. This could be of importance since NO modulates several other tran-scription factors, including NF- B [97, 98], activating tran-scription factor 3 (ATF3) [99], and potentially Sp1 [87, 100];

Table 2. Effect of Gestational Diabetes, Hyperglycaemia and Insulin on hENT1-Mediated Adenosine Transport and Extracellular

Concentration in HUVEC

Transporters x106/Cell Molecules/Cell/sec Adenosine ( M)

Normal pregnancies

5 mM D-Glucose 1.8 ± 0.1* 356 ± 30 0.051 ± 0.011*

5 mM D-Glucose + 1 nM Insulin 1.1 ± 0.1 372 ± 42 1.82 ± 0.02

25 mM D-Glucose 0.7 ± 0.2 324 ± 45 2.21 ± 0.4

25 mM D-Glucose + 1 nM Insulin 0.8 ± 0.3 353 ± 37 1.91 ± 0.3

Gestational diabetes

5 mM D-Glucose 0.9 ± 0.2 323 ± 15 1.3 ± 0.2

5 mM D-Glucose + 1 nM Insulin 0.7 ± 0.1 313 ± 17 1.9 ± 0.4

25 mM D-Glucose 0.8 ± 0.3 335 ± 22 1.6 ± 0.3

25 mM D-Glucose + 1 nM Insulin 0.8 ± 0.2 343 ± 12 1.4 ± 0.3

HUVEC isolated from normal pregnancies or pregnancies with gestational diabetes were cultured in the presence of 5 or 25 mM D-glucose for 24 hours. Insulin was added for the last 8 hours of the D-glucose incubation period (see [19, 141]). Number of transporters per cell (NT) was estimated from the maximal specific nitrobenzylthioinosine (NBTI) covalent

binding (Bmax) values by the expression NT =Bmax

106 • N where N is Avogadro’s number and Bmax is expressed in pmol/106 cells [2, 3, 14, 80], turnover of adenosine (i.e. number of

molecules per cell per second) was estimated by the ratio for maximal velocity (Vmax) for adenosine transport and Bmax (i.e. Vmax/Bmax) [2, 3, 14, 80] and extracellular adenosine con-

centration by h.p.l.c. as described [18]. Values are mean ± S.E.M. (n=12-25). *P<0.05 vs. all other corresponding values.

Endothelial Nucleoside Transporters in Diabetes Current Vascular Pharmacology, 2009, Vol. 7, No. 4 441

however, the exact molecular evidences of how NO is acting as an activator of hCHOP-C/EBP and/or Sp1 in HUVEC repressing the human SLC29A1/hENT1 promoter expression is still under study (M Farías, C Puebla & L Sobrevia, un-published data). As a summary, most mechanisms regarding regulation of hENT1 expression and activity have been de-scribed in HUVEC from gestational diabetes are described in Fig. (1).

HYPERGLYCAEMIA

Many studies suggest that post-prandial hyperglycaemic excursions also adversely affect the vascular wall [101-103]. Therefore, an optimal short- and long-term glycaemic con-trol is required to decrease the risk of vascular complications in diabetic patients [104]. Hyperglycaemia-induced oxidative stress in vascular endothelial cell is produced by two inde-pendent mechanisms: (1) metabolic production of reactive oxygen species (ROS) due to disruption of mitochondrial function and electron leakage, NADH overload, increased polyol and glucose-amine pathways flux, methylglyoxal and glyoxal production, activation of NADPH oxidase, diacyl-glycerol-dependent activation of conventional and novel PKC isoenzymes or inhibition of the thioredoxin system; (2)

glycoxidation and non-enzymatic glycation of proteins lead to an excessive production of free radicals [105, 106].

Interestingly, vascular endothelial cells maintained at 5.5 or 23 mM D-glucose up- or down-regulated their rates of hexose transport, respectively [107], a phenomenon that could result from D-glucose-induced destabilization of GLUT-1 mRNA and reduced GLUT-1 protein content and availability at the plasma membrane [108, 109]. The glucose derived metabolites and/or other molecules that have the potential of acting as intracellular signals to operate this auto-regulatory mechanism in this type of endothelium have not been identified yet [3, 6]. However, it is clear that a criti-cal role resulting from the combination of hyperglycaemia and an intense oxidative stress in the development of endo-thelial cell dysfunction could be associated with diseases where D-glucose metabolism is altered [107]. For example, in case of glycogen synthesis, long-term incubation of mac-rovascular endothelial cells with high glucose (30 mM) per se increased glycogen synthesis and glycogen content [110]. This effect in high glucose-exposed endothelial cells could help to diminish or to prevent intracellular glucose accumu-lation, a phenomenon that damages tissues and cells that take up glucose in an insulin-independent manner [111].

Fig. (1). Effect of gestational diabetes on hENT1 expression and activity in HUVEC. Gestational diabetes is associated with reduced ( )

hENT1-mediated adenosine transport in human umbilical vein endothelial cells (HUVEC). This phenomenon could be explained by low

protein abundance in the plasma membrane, probably due to reduced transcriptional activity of SLC29A1 gene (for hENT1) in HUVEC

from gestational diabetes. A change in the recycling kinetics of hENT1 (from and to the plasma membrane), redirecting functional transport-

ers (hENT1) to intracellular compartments could contribute to hENT1 functional alteration. The increase in the expression and activity of

transcription factors with repressive activity (FT-) such as hCHOP and C/EBP (hCHOP-C/EBP ) is critical in reducing the transcriptional

activity of SLC29A1 promoter in HUVEC from gestational diabetes. This repression depends in part on increased ( ) NO synthesis in this

cell type compared with HUVEC from normal pregnancies. Eventually, the participation of protein kinase C (PKC) by signaling mechanisms

still unclarified (?) is feasible. On the other hand, both PKC and mitogen-activated protein kinases p42 and p44 (p42/44mapk

) could contribute

to activate SLC29A1 promoter via transcription factors with induction activity (FT+), such as specific protein 1 (Sp1), in regions near the

translation initiation site (ATG). Apparently, this potential stimulatory effect is masked by the repressive action of FT(-). All these events

may lead to increased extracellular adenosine (Ado) concentration in HUVEC isolated from pregnancies with gestational diabetes. L-Arg: L-

arginine. CATs: cationic aminoacid transporters. eNOS: endothelial NO synthase. L-Cit: L-citrulline (from data in [89]).

442 Current Vascular Pharmacology, 2009, Vol. 7, No. 4 Westermeier et al.

D-Glucose Metabolism in Endothelium

Glycolysis (i.e. intracellular gradual degradation of D-glucose and other simple sugars) is basically an anaerobic process whose main steps occur without oxygen consump-tion. The first two essential steps in glycolysis are the trans-port of D-glucose through the plasma membrane and subse-quent metabolism initiated by phosphorylation at expenses of hexokinase activity. There are reports indicating that D-glucose is actively metabolized in endothelial cells [112]. In the endothelium of rat coronary microvasculature more than 98% of incorporated D-glucose is metabolized to lactate [113]. Under a condition of a normal concentration of D-glucose ( 5 mM), the contribution of the hexose monophos-phate pathway accounts for ~1% of D-glucose metabolism and the Krebs cycle for only ~0.04%, suggesting that in this type of endothelial cells almost all of the energy obtained from D-glucose catabolism is generated glycolytically [3]. At lower D-glucose concentrations (~1 mM) oxidation of D-glucose via the Krebs cycle is higher. This oxidative metabo-lism in endothelial cells is inhibited in normal concentrations of D-glucose, demonstrating that endothelial cells express the Crabtree effect (i.e. an inhibitory effect of D-glucose on mitochondrial respiration) [113]. Endothelial cells synthesize ATP primarily via glycolysis with a relatively low O2 con-sumption [114-116]. Thus, these endothelial cells are able to withstand prolonged periods of substrate deprivation and can adapt to hypoxia due to their low energy demand and high glycolytic activity [115, 117, 118].

The effect of an extracelular concentration of D-glucose over the physiological level in endothelial cells is usually specific to the type of endothelial cells i.e. micro- or mac-rovascular, and according with the vascular bed origin [119-122]. The cytosol of endothelial cells is reduced by accumu-lation of NADH and transformation of pyruvic acid to lac-tate, as described in microvascular endothelial cells from bovine corpus cavernosum [114] and rabbit brain microves-sels [123]. In some endothelial cell types, the polyol pathway reduces D-glucose to sorbitol via aldose reductase (AR), which has an extremely low affinity for D-glucose (Km 100 mM), but is activated by D-glucose itself or D-glucose-6-phosphate [104]. Conversion of D-glucose to sorbitol by aldose reductase forms NADP

+ and may compete with other

NADPH-requiring reactions such as conversion of oxidized glutathione (GSSG) to its reduced form, reduced glutathione (GSH) [124, 125]. On the other hand, there are studies show-ing that activation of the polyol pathway in endothelial cells may not be directly responsible for the associated decrease in NADPH content [125]. Studies in primary cultures of HUVEC suggest that hyperglycemia is associated with an increase in the synthesis of glycogen and storage [110]. This phenomenon has been proposed as a cellular mechanism to decrease the intracellular accumulation of D-glucose protect-ing the endothelium of the well-known deleterious effects of this hexose in this cell type. These authors also suggest that glycogen metabolism is regulated by the availability of D-glucose and levels of reserves of glycogen in human endo-thelial cells. Moreover, according to these authors total transport of D-glucose does not depend on the extracellular level of this hexose. Unfortunately, membrane transport was not addressed under experimental conditions designed to

detect initial velocity of uptake limiting the interference of D-glucose metabolism in this phenomenon [110].

Regulation of Transport and Metabolism of D-Glucose in

Endothelium

There is experimental evidence in rats showing that D-glucose metabolism depends on the activity of GLUT1 and that these phenomena could play a role in the regulation of endothelial cell function [126]. Thus, D-glucose transport could be a key stage in trigger intracellular mechanisms regulating endothelial function. Transport of D-glucose in the endothelium is modulated by various actors, including NO, adenosine, insulin, and physiopathological conditions such as diabetes mellitus, obesity, intrauterine growth restric-tion [3, 127]. Recent studies show that NO regulates trans-port and potentially metabolism of D-glucose in endothelial cells from HUVEC. Sodium nitroprusside (SNP, a spontane-ous NO donor) increases GLUT1 expression and the hexokinase activity, both mechanisms that seem to depend of the activity of the intracellular signalling molecules PKC and PI3K [128]. It has also been reported that lipopolysaccharide (LPS) increases GLUT1 protein abundance in liver endothe-lial cells in fasting rats, suggesting that LPS-induced NO synthesis in this cell type may be responsible for the effect on the expression of this type of D-glucose transporters [129]. Other studies show that NO would be a modulator of D-glucose transport by increasing it by about 4-5 times the basal transport in response to SNP in rat skeletal muscle [130]. It has recently been reported that in primary cultures of HUVEC insulin does not regulate total transport of D-glucose [110], this could suggest lack of expression, or at least activity of GLUT4 in this cell type. These data are con-tradictory with earlier findings showing that, in the same cell type, insulin induces uptake of 3-o-methyl-D-glucose (3oMG) [131]. Preliminary results suggest that GLUT4 is expressed in HUVEC and that insulin induces an apparent redistribution of this transporter (C Puebla, L Sobrevia, un-published data). The fact that D-glucose transport in HUVEC is potentially independent of insulin, dos not ruled out the presence of GLUT4. Similarly, this type of D-glucose transporter could potentially be regulated by other sort of stimuli such as NO [132] or adenosine [3].

In bovine retinal endothelial cells (BREC) IGF-1 pro-duces a rapid increase in MAP kinase activity and subse-quent phosphorylation of ERKs [133, 134]. In cytotro-phoblasts, a cell type that in terms of mother-to-fetus vecto-rial transport of various metabolic substrates, including D-glucose, would be functionally coupled to the microvascular endothelial function in the human placenta [26, 127], it has been reported that resistin modulates of GLUT1 expression and activity, a mechanism that involves ERK1/2 activation [135]. This dependency of ERKs activation for the cellular effects of D-glucose has also been reported in HUVEC [14], among other endothelial cells types [6]. For example, in pri-mary cultures of mouse aortic endothelial cells it has been reported that exposure to a culture medium containing a high D-glucose (~22 mM) increases ERK1 (~2.6 times) and ERK2 (~1.8 times) phosphorylation. This phenomenon has been proposed as a mechanism responsible for permeability increase and proliferation of aortic endothelial cells in diabe-tes mellitus [136].

Endothelial Nucleoside Transporters in Diabetes Current Vascular Pharmacology, 2009, Vol. 7, No. 4 443

Effect of D-Glucose on Nucleoside Transport

An elevated extracellular D-glucose concentration also induces changes in the transport properties of vasoactive metabolic substrates by the endothelium such as the endoge-nous nucleoside adenosine [14, 19, 27, 86, 87] (Table 3). In a hyperglycaemic environment it has been reported a reduced adenosine transport leading to extracellular accumulation of this nucleoside [19, 27, 87]. Apparently these changes are due, almost exclusively, to a reduced hENT1-mediated adenosine transport associated with reduced number of hENT1 membrane transporters available at the plasma mem-brane, as well as a reduced number of copies of hENT1 mRNA [19, 27, 87]. It has been shown that L-arginine trans-port and NO synthesis is stimulated by adenosine [2, 5-7, 22, 29, 67, 70, 137]. Similarly, in HUVEC from normal preg-nancies incubated with nucleoside transport inhibitors (for example nitrobenzylthioinosine, NBTI) an increased activity of L-arginine/NO signaling pathway has been reported [8]. This effect of NBTI was associated with a higher number of copies for hCAT-1 mRNA (high-affinity cationic amino acid transporter for L-arginine), along with increased eNOS activ-ity and expression (mRNA and protein) [8, 78]. The fact that this nucleoside is recognized in HUVEC as a modulator of the activity of L-arginina/NO signaling pathway, suggests that changes in extracellular adenosine levels induced by high extracellular D-glucose [19] are crucial in pathologies

associated with endothelial dysfunction resulting from ad-verse environmental conditions which result in states of hy-perglycemia such as diabetes mellitus, including gestational diabetes [21].

Although there are several studies demonstrating func-tional alterations induced by hyperglycaemia, it is unknown which intermediary metabolites of glycolysis are responsible for the phenotypic changes of the endothelium at this adverse environmental condition [3, 6, 11]. Surprisingly, nothing is known in the literature regarding the possibility that changes in L-arginine or adenosine transport may result from phe-nomenon of altered D-glucose membrane transport, and whether this is a time-limiting step for the effect of this hexose in endothelial function [3, 6, 11]. It has also been reported that glycolysis and excitation-contraction mecha-nisms are functionally coupled through the capture or release of Ca

+2 from sarcoplasmic reticulum [138]. This phenome-

non involves analogues of intermediary metabolites and in-hibitors of D-glucose metabolism, such as 2-deoxy-D-glucose (2-DG) and iodoacetate (IAA), and D-glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), fructose-1,6-byphos-phate (FBP), phosphoenolpiruvate (PEP) or the ter-minal glycolysis products piruvate or L-lactate. Some of these metabolites increase the activity of ryanodine receptors (RyRs), such as F6P and FBP, however piruvate reduced opening probability of RyR [138]. Thus, diferente metabo-

Table 3. Effect of High D-Glucose on Nucleoside Transporters

Nucleoside

Transporter

Tissue Transport Activity mRNA Level Protein Abundance References

CNT1 Rat fibroblasts Unaltered Unaltered – [84]

Rat fibroblasts Unaltered Unaltered – [84]

Rat T lymphocytes – Unaltered – [170] CNT2

Rat B lymphocytes – Unaltered – [167]

HUVEC Reduced Reduced Reduced [19, 27, 87]

HUVEC Reduced Reduced – [86]

Rat fibroblasts Reduced Reduced – [84]

Rat fibroblasts Reduced Reduced Reduced [16]

Rat T lymphocytes Reduced Reduced – [170, 171]

Rat B lymphocytes Reduced Reduced – [167]

ENT1

HASMC Increased Increased Increased [172]

HUVEC Unaltered Unaltered Unaltered [19, 27]

Rat fibroblasts – Unaltered – [84]

Rat T lymphocytes Unaltered Unaltered – [170]

Rat T lymphocytes Unaltered – – [171]

Rat B lymphocytes – Unaltered – [167]

ENT2

HASMC – Increased – [172]

The effect of high D-glucose on transport activity, protein abundance and mRNA level or number of copies of mRNA for concentrative (CNTs) and equilibrative (ENTs) nucleoside

transporters. HUVEC: human umbilical vein endothelial cells (primary cultures). HASMC: human aortic smooth muscle cells (primary cultures). –: not reported.

444 Current Vascular Pharmacology, 2009, Vol. 7, No. 4 Westermeier et al.

lites derived from D-glucose metabolism have diferente ef-fects as regulatory molecules in biochemical phenomena.

Regulation of Transcription Factors by D-Glucose

Although the intrinsic transcription and translation regu-latory mechanisms activated by D-glucose in vascular cells have not been described, it is clear the impact produced by hyperglycemia in the regulation of gene expression in mam-malian cells [139]. Most of what we know about the tran-scriptional mechanisms used by D-glucose to regulate gene expression comes from experiments that used specialized cells or tissues that accumulate and store D-glucose or se-crete insulin in response to D-glucose, such as hepatocytes, skeletal muscle, adipose tissue and pancreatic islet -cells. In contrast, D-glucose regulation of vascular gene seems to be largely independent of insulin [139], although this hormone regulates gene expression and cellular function in not ca-nonical tissues for its range of biological action [140]. For example, in HUVEC insulin reverses the stimulatory effect of high D-glucose on hCAT1-mediated L-arginine transport, NO synthesis and eNOS expression [141], as well as insulin reverses the inhibitory effect of hyperglycaemia (and gesta-tional diabetes) on hENT1-mediated adenosine transport and increases hENT2 activity and expression in HUVEC [19] (Table 2).

D-Glucose modulates the transcriptional activity of vari-ous genes by activating several transcription factors, such the zinc finger promoter-selective transcription factor specific protein 1 (Sp1)[142], upstream stimulatory factors (USF) [142], carbohydrate response element binding factor (ChREBP) [143-145], sterol regulatory element binding pro-tein-1 (SREBP-1) [146, 147], early growth response gene-1 (Egr-1) [148], NF- B [149, 150], among others. In general, the promoter elements flanked by these transcription factors consist of E-boxes and GC-rich elements, as for Sp1 [139]. In turn, GC-rich regions can be recognized by transcription factors that potentially interact with Sp1, such as ZBP-89 [151]. It has also been reported that metabolites derived from glycolysis could trigger signalling pathways that control the expression of certain genes. G6P induces synthesis of fatty acid synthase (FAS) mRNA in cultures of adipocytes [152]. In adipose tissue it has been reported a positive regulation by D-glucose on the synthesis of the enzyme that catalyzes the degradation of triacylglycerol, hormone-sensitive lipase (HSL) which contains an E-box on its promoter and requires the involvement of a D-glucose metabolite [153].

In studies regarding the regulation of hENT1 expression, the analysis of the sequence of -1114 bp from the ATG (tran-scription initiation site) of the promoter region of SLC29A1 shows the presence of at least one responsive element to Sp1 (using MatInspector,

©Genomatix Software, GmbH, and

MOTIF, http://motif.genome.jp, with a cut-off of 80) [87, 100]. This region has been suggested to be involved in the reduction of SLC29A1 (hENT1) expression in a NO-dependent manner [87, 100], as proposed in HUVEC from gestational diabetic pregnancies [21]. Thus, it is possible that D-glucose transport (potentially via GLUT1 and/or GLUT4 in human fetal endothelial cells), and metabolites derived from glycolysis could be responsible for the inhibition of hENT1-mediated adenosine transport and expression of

SLC29A1 via a mechanism that depends on NO-dependent Sp1 [87] and/or ZBP-89 [100] in HUVEC (see Fig. 2).

Regulation of SLC29A1 Gene

SLC29A1 gene expression regulation by transcription factors has been studied in hypoxia where there is an attenu-ated expression of both hENT1 as hENT2, which has been explained by a negative regulation by the hypoxia inducible factor 1 (HIF-1) [96]. In hypoxia the relationship between HIF-1 activity and gene expression is seen as a decrease in the mRNA level for ENT1 and ENT2 and a lower promoter transcriptional activity (-344 to -756 bp from ATG), which has two possible binding sites of HIF-1 in different endothe-lial cell lines [96]. Other transcription factors that may be involved in mouse ENT1 (mENT1) regulation are Sp1 and Myc-associated zinc finger protein (MAZ), suggesting that these factors could be responsible for basal promoter activity [154]. Several studies also show that NO modulates gene expression [21, 155, 156] through the activation or inhibition of transcription factors, such as Sp1, NF B, c-Myc y AP-1 [155, 157-160]. It has been reported that NO reduces Sp1 binding to promoter of PKG-I gene in bovine aortic smooth muscle cells [161] and the promoter of tumor necrosis factor

(TNF- ) in U937 cells [160]. Moreover, it should be noted that Sp1 could interact with other transcription factors, such as ZBP-89 [98], which is capable of achieving suppression of the transcriptional activity of the gene coding for vimentin protein, even in the presence of Sp1, which lead to activation of this gene [151]. On the other hand it has been reported that ZBP-89 negatively regulates the expression of growth hormone receptor in response to saturated fatty acids, which may explain the altered activation due to abnormal interac-tion between this hormone and its receptor associated with diabetes mellitus [162]. Interestingly, the analysis of the se-quence for -1114 bp of SLC29A1 promoter region (from ATG) shows the existence of a Sp1 response element, sug-gesting the possibility that this site could be involved in the NO-dependent reduced hENT1 expression recently reported in primary cultures of HUVEC from gestational diabetes pregnancy [21]. In turn, the presence of a ZBP-89 binding site in the SLC29A1 promoter region, might suggests interac-tion of ZBP-89 with Sp1 in HUVEC leading to reduced ex-pression of hENT1 (Fig. 2), as previously reported for re-pression of the expression of other genes in mammalian cells [163-165].

CONCLUDING REMARKS

Equilibrative nucleoside (ENTs) plasma membrane transporters are crucial mechanisms to maintain a normal function of endothelium in the foetus-placental unit. ENTs are in charge of regulating extracellular levels of nucleo-sides, mainly adenosine, thus modulating its vascular effects. One of the consequences of the abnormal removal of ex-tracellular adenosine by human foetal endothelium in gesta-tional diabetes is the activation (via purinoceptors A2A) of L-arginine transport and synthesis of NO (i.e. L-arginine/NO pathway). This mechanism results in decreased expression of hENT1 due to elevated NO activating transcription factors involved in the modulation of the expression of SLC29A1 gene under this pathology. Interestingly, elevated levels of extracellular D-glucose (an environmental condition to

Endothelial Nucleoside Transporters in Diabetes Current Vascular Pharmacology, 2009, Vol. 7, No. 4 445

which the foetal vessels could be exposed in gestational dia-betes), induce similar alterations in foetal endothelial func-tion. Despite the fact that NO synthesis and/or bioactivity is altered in diabetes mellitus types 1 and 2, and in gestational diabetes, an evident lack of information regarding the poten-tial involvement of hENTs in the human feto-placental unit in diabetes mellitus makes hard to understand these phenom-ena at a cellular and molecular level, which could contribute to the aetiology of pregnancy diseases in diabetes and even-tually program the foetus to adult disease.

ACKNOWLEDGEMENTS

Supported by Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT 1070865, 1080534, 7080139, 7070249) and Comisión Nacional de Ciencia y Tecnología (CONICYT 24071039, 23070213), Chile. M. Farías holds a CONICYT- and School of Medicine, Pontificia Universidad Católica de Chile- PhD fellowships (Chile). C. Escudero holds a MECESUP- and School of Medicine, Pontificia Uni-versidad Católica de Chile-PhD fellowships (Chile). C. Pue-bla and J.L. Vega hold CONICYT PhD fellowships (Chile). F. Westermeier was recipient of a Pontificia Universidad Católica de Chile-PhD fellowship (Chile) and holds a CONICYT PhD fellowship. We thank the researchers at the Cellular and Molecular Physiology Laboratory (CMPL) and Perinatology Research Laboratory (PRL) of the Pontificia Universidad Católica de Chile (PUC) for their contribution in the production of the experimental data that has been cited throughout the text. Authors also thank Mrs. Jesenia Acurio for excellent technical assistance, and the personnel of the Hospital Clínico Pontificia Universidad Católica de Chile labour ward for supply of placentas.

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Received: September 31, 2008 Revised: November 07, 2008 Accepted: January 08, 2009

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