Current Vascular Pharmacology, 2009, 7, 475-485 475

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

Potential Cell Signalling Mechanisms Involved in Differential Placental Angiogenesis in Mild and Severe Pre-Eclampsia

Carlos Escudero1,2,*, Carlos Puebla

2, Francisco Westermeier

2 and Luis Sobrevia

2

1Department of Basic Sciences, Faculty of Sciences, Universidad del Bío-Bío, Campus Fernando May, Chillán, Chile;

2Cellular 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 Uni-

versidad Católica de Chile, P.O. Box 114-D, Santiago, Chile

Abstract: Fetal and neonatal morbidity and mortality is high in severe pre-eclampsia compared with mild pre-eclampsia

and normotensive pregnancies. Causes for these fetal disturbances had been associated with iatrogenic prematurity and re-

duction in placental blood flow. Actual evidences suggest that in severe (early-onset) pre-eclampsia a reduction in placen-

tal angiogenesis could be a mechanism responsible for the reduced placental blood flow, while in mild (late-onset) pre-

eclampsia normal placental blood flow could result from either no alteration or increased placental angiogenesis, or re-

duced vessel resistance. Since adenosine is involved in endothelium proliferation and angiogenesis, and umbilical and ma-

ternal blood level of this nucleoside is elevated in pre-eclampsia compared with normal pregnancies, it is feasible that pla-

cental angiogenesis in mild and/or severe pre-eclampsia involves adenosine-dependent cell signaling mechanisms. There

are not reports regarding adenosine role in placental angiogenesis neither in normal nor in pathological pregnancies. How-

ever, it is well established that adenosine stimulates adenosine receptors triggering expression of angiogenic factors such

as vascular endothelial growth factor (VEGF). VEGF stimulates VEGF receptors type 1 and 2, activating signaling cas-

cades that involve increased synthesis of endothelial-derived nitric oxide (NO). On the other hand, the soluble VEGF re-

ceptor type 1 (sFlt-1), whose plasma concentration is increased in severe compared with mild pre-eclampsia, reduces an-

giogenesis, spotting sFlt-1 as a factor that could potentially be involved in this phenomenon. This review focuses on the

available evidence regarding a potential differential mechanism of placental angiogenesis in mild compared with severe

pre-eclampsia, and analyzes the potential role of adenosine/VEGF/VEGF receptors/NO signaling cascade in this phe-

nomenon.

Keywords: Angiogenesis, nitric oxide, endothelium, pre-eclampsia.

INTRODUCTION

Pre-eclampsia refers to several vascular alterations char-acterized by maternal hypertension and proteinuria [1, 2] affecting 5-10% of the pregnancies worldwide [3, 4]. This pathology is recognized as the principal cause of maternal morbidity and mortality and fetal metabolic disturbances [3, 4]. Pre-eclampsia is divided in mild or ‘late-onset’ pre-eclampsia and severe or ‘early-onset’ pre-eclampsia, [4] a classification that allows identifying patients with high risk of maternal or fetal complications during pregnancy [5-9]. In this sense, morbidity and mortality of the fetus and the neo-nate from women with severe pre-eclampsia are higher than normotensive pregnancies, and when compared with the morbidity and mortality incidence in mild pre-eclampsia [3-10]. Etiology of fetal complications in pre-eclampsia is con-troversial, but had been associated with iatrogenic prematur-ity and reduction in placental blood flow [11-15].

Actual

evidences suggest that in severe pre-eclampsia a reduction in placental angiogenesis could be a mechanism responsible for the observed reduced placental blood flow, while in mild

*Address correspondence to this author at the Department of Basic Sci-

ences, Faculty of Sciences, Universidad del Bío-Bío, Campus Fernando May, Chillán, Chile; Tel: 56-42-253256; Fax: 56-42-253246;

E-mail: cescudero@ubiobio.cl

pre-eclampsia the observed normal blood flow could result from either no alterations or increased placental angiogene-sis, or reduced vessel resistance [11, 12].

Adenosine is an endogenous purinergic nucleoside in-volved in angiogenesis and the modulation of vascular tone [16-18]. Interestingly, umbilical and maternal blood levels of adenosine are higher in mild and severe pre-eclampsia com-pared with normal pregnancies [19-26], a phenomenon that could result in altered biological effects of this nucleoside in the vasculature of the mother and the placenta. Adenosine stimulates endothelial cell proliferation and migration via activation of adenosine A2A and/or A2B receptors involving increased expression of angiogenic factors, including the vascular endothelial growth factor (VEGF) [27-30]. VEGF is the unique mitogen that acts specifically in endothelial cells by activating VEGF membrane receptors (VEGFR) [31-33]. Activation of VEGFR triggers intracellular signalling path-ways that involve increased nitric oxide (NO) synthesis [34-36], a gas that seems to be determinant in endothelial cell proliferation in the fetal-placental unit [37]. However, there are not reports regarding the proangiogenic role of NO in this vascular bed in pre-eclampsia [36, 37]. Interestingly, the plasma level of the soluble VEGF type 1 receptor (sFlt-1) in the mother and the fetus is higher in severe pre-eclampsia compared with mild pre-eclampsia or normal pregnancies

476 Current Vascular Pharmacology, 2009, Vol. 7, No. 4 Escudero et al.

[38, 39]. Since sFlt-1 is a factor that reduces angiogenesis, it could also be involved in reduced placental angiogenesis characteristic of severe pre-eclampsia.

In this review we focus on placental angiogenesis and the potential molecular mechanisms determining these phenom-ena in severe and mild pre-eclampsia. We discuss informa-tion regarding the proangiogenic effect of adenosine via adenosine receptors leading to synthesis and release of VEGF by the endothelium from the fetal-placental unit. In-sights of the mechanisms accounting for these effects in the macro (umbilical vessels) and microvasculature (chorionic circulation) will be contrasted. A general review and discus-sion of the available hypothesis regarding fetal outcome in pre-eclampsia i.e. vascular tone and angiogenesis regulation, will be given.

PRE-ECLAMPSIA

Pre-eclampsia is a human syndrome that is recognized from the 20

th week of gestation and is characterized by ma-

ternal hypertension and proteinuria [2, 4] as defined by the

International Society for the Study of Hypertension in Preg-nancy (ISSHP) [1]. Pre-eclampsia affects between 5-10% of pregnancies and is the primary cause of maternal death, with high fetal death prevalence [1-10, 40-42]. Maternal pre-eclampsia is probably more than one unique disease, but instead it is a syndrome, with major differences between mild or late-onset pre-eclampsia, without demonstrable fetal involvement, and severe or early-onset pre-eclampsia, which is associated with low birth weight and preterm delivery [3]. Dissociation between mild and severe pre-eclampsia is am-biguous, but in general mild pre-eclampsia (>34 weeks of gestation) is defined by blood pressure 140/90 mmHg and proteinuria 3 g/24 h, whereas severe pre-eclampsia (<34 weeks of gestation) is defined by blood pressure 160/110 mm Hg and proteinuria 5 g/24 h [1, 43]. This classification of pre-eclampsia is determinant since allows the identifica-tion of patients with high risk of maternal or fetal complica-tions, is essential to select an appropriate clinical manage-ment of the disease, and potentially, it could be important to predict the fetal condition after pregnancy or even in the adulthood [5-9]. Thus, severe pre-eclampsia is associated with almost 20-fold more risk for maternal death [44],

4-fold

for recurrence in the second pregnancy [45] and 3-fold for cardiovascular disease risk late in the adult life of women [46, 47].

The aetiology of pre-eclampsia is unclear; however, the actual widely accepted hypothesis suggests that shallow tro-phoblast invasion to spiral maternal vessels, avoid maternal vessels transformation from resistance to capacitance vessels [8, 9, 48]. This phenomenon induces a reduction in placental blood flow, which is associated with hypoxia and low nutri-ent uptake in fetal-placental tissues [3, 15]. These effects of pre-eclampsia lead to over-expression of anti-angiogenic proteins such as sFlt-1. This soluble receptor binds VEGF and placental growth factor (PlGF) in the maternal circula-tion preventing activation of their membrane receptors. This phenomenon results in blockage of VEGF angiogenic and vasodilatory effects, thus causing vasoconstriction (hyper-tension) and glomerular endotheliosis (proteinuria). At the same time, low placental blood flow induces cytotrophoblast

apoptosis, and increases free radical generation and pro-inflammatory cytokines synthesis, molecules that are re-leased to the maternal circulation contributing to maternal endothelial dysfunction, generation of a prothrombotic state and systemic damage characteristic of this pathology [7-9, 48]. All these phenomena in fact could be the result or lead-ing to the generation of a potential placental adaptation in response to the maternal unfavorable environment in pre-eclampsia.

Fetal Outcome During Pre-Eclamptic Pregnancies

Fetal outcomes during pre-eclamptic pregnancies depend of several factors such as, gestational age at delivery, sever-ity of the disease, clinical management and co-morbidity [3, 10]. In a secondary analysis of data collected in the World Health Organization (WHO) Antenatal Care Trial, fetal and neonatal mortality in pre-eclampsia resulted to be ~2.2 and 2.4%, respectively [6]. This Trial also showed that the need of intensive care unit is higher in newborns from pre-eclamptic pregnancies compared with newborns from normal pregnancies. Additionally, diagnosis of intrauterine growth restriction (IUGR, weight under 10

th percentile for gesta-

tional age) is more frequently associated with pre-eclampsia compared with idiopathic IUGR, [5, 6]

and severe rather than

mild pre-eclampsia is often associated to IUGR [5, 49, 50] and fetal death [10]. Moreover, the life expectancy in new-borns >27 weeks of gestation or fetal weight >600 g is more than 50% in pre-eclamptic pregnancies [51]. Additionally, newborns from severe pre-eclampsia exhibit high blood pressure compared with normal pregnancies [52]. Interest-ingly, when IUGR, pre-eclampsia and preterm delivery (<37 weeks of gestation) were all together considered in the analysis, the risk for cardiovascular diseases reached 16-fold compared with fetuses with adequate weight for gestational age, non hypertensive pregnancies and term deliveries [53]. In relation to gender, daughter from pre-eclamptic pregnan-cies have high risk (2-fold) of developing severe pre-eclampsia in their pregnancies [54] and low risk (4-fold) for breast cancer [55, 56]. The latter findings related with fetal outcome in pathological pregnancies, included pre-eclampsia, have been a focus of interest in the potential asso-ciation of pregnancy diseases with programming hypothesis [57, 58] (see Table 1). All together these observations sug-gest that mild and severe pre-eclampsia could be different pathologies. In fact it is feasible that in mild pre-eclampsia the fetus is adapted to the pathological condition; however, in severe pre-eclampsia even when fetal adaptation to the pathology occurs, this phenomenon is abnormal [7].

CURRENT HYPOTHESIS FOR FETAL OUTCOME IN

PRE-ECLAMPSIA

Placental Vascular Tone Regulation in Pre-Eclampsia

Since placental tissue lack innervation [59] local regula-tion of the feto-placental vasculature depends on a careful equilibrium between the synthesis, release and bioavailabil-ity of vasoconstrictors and vasodilators [36, 37]. Reduction in placental blood flow has been associated with increased sensitivity of the placental vasculature to vasoconstrictors, such as angiotensin II, endothelin 1 and 3, prostaglandins F2 (PGF2 ), PGE2 and PGD2, and thromboxane A2 [60, 61].

Placenta Angiogenesis in Pre-Eclampsia Current Vascular Pharmacology, 2009, Vol. 7, No. 4 477

Table 1. Fetal and Placental Outcomes in Pre-Eclampsia

Mild Severe Outcome

Pre-Eclampsia Pre-Eclampsia

References

Foetus

Delivery <37 weeks 18 % 58 % [5]

Delivery <35 weeks 10 % 36 % [5]

Weight <10th percentile (SGA) 5 % 11 % [5]

Weight <2500 g 11 % 37 % [50]

Weight at <34 weeks (g)* -46 to -245 -552 to -665 [49]

Weight >90th percentile (LGA) 18 % 4 % [5]

Admission to NICU 24-27 % 38-43 % [5,50]

RDS 5 % 17 % [5,50]

Fetal death 0-0.2 % 1-7 % [5,50]

Neonatal mortality 0-0.8 % 2 % [5, 50, 161]

Foetus-placenta circulation

Placental weight (g) 463 253 [12]

Terminal capillary parameters†

Length (cm) 148 58 [12]

Surface area (m2) 5.6 1.8 [12]

Volume (m3) 36 17 [12]

High vascular impedance** 31 % 57 % [162]

Umbilical VEGF (pg/ml) ‡ 356 625 [158,159]

sFlt-1 (ng/ml)‡ 8.1 94.9 [159]

Fetal weight estimation using different parameters: small of gestational age (SGA), large for gestational age (LGA), weight <2500 g or delta ( ) weight compared with controls ac-

cording with gestational age. NICU, neonatal intensive care unit. RDS, respiratory distress syndrome. *Study performed in different gestational ages since 32 weeks up to 41 weeks

of gestation. Average using 34 weeks as a cut-off is shown. † Mean data for quantification performed in tissue analysis. Variables included are related with placental capillary forma-

tion. **Impedance measured by echography as an indicator of vascular resistance. ‡Vascular endothelial growth factor (VEGF) and soluble VEGF receptor type 1 (s-Flt1) measured in

the human umbilical plasma. Pre-eclampsia associated with intrauterine growth restriction (IUGR) was considered as severe pre-eclampsia, whereas pre-eclampsia without IUGR was

considered as mild pre-eclampsia.

Additionally, a reduced activation of ATP-sensitive potas-sium channels (KATP), which are expressed and functional in the human feto-placental unit [62, 63], has been proposed to play a role in the impaired relaxation of human umbilical artery smooth muscle in pre-eclampsia [64]. The human pla-cental vasculature is also highly sensitive to vasodilators, such as prostacyclin, PGE1, NO and nitroglycerin [60, 65].

Additionally, the endogenous nucleoside adenosine also acts as modulator of vascular tone inducing vasodilatation or vasoconstriction [66-68] or altering the effect of vasconstric-tors [69] depending on the flanked vascular feto-placental bed. Endothelium-derived NO is an important modulator of placental blood flow, and altered synthesis, bioavailability and/or biological actions of NO has been associated with abnormal blood flow in pre-eclampsia, IUGR and gestational diabetes [26, 36, 37]. Interestingly, echographic studies show a reduction in umbilical artery blood flow estimated from an

increased pulsatility index [14] or increased number of pla-centa tissue areas without blood flow (hypoechogenic) [70] in severe pre-eclampsia. However, in mild pre-eclampsia no significant differences in umbilical artery pulsatility index compared to normal pregnancies have been reported [71]. Since placental tissue in pre-eclampsia is associated with hypoxia [72] and because hypoxia is an important inductor for vascular development, [73] it is feasible that this abnor-mal environmental condition is associated with increased formation of new vessels in this disease. However, there are not studies addressing oxygen and/or hypoxia as markers for pre-eclampsia severity, but it has been suggested that the magnitude of hypoxia could be different in mild and severe pre-eclamptic pregnancies [11, 74, 75]. Thus, a differential regulatory mechanism, perhaps dependent on oxygen level, could be acting for placental vessel formation in mild or se-vere pre-eclampsia [12].

478 Current Vascular Pharmacology, 2009, Vol. 7, No. 4 Escudero et al.

Placenta Angiogenesis in Pre-Eclampsia

Angiogenesis is the formation of new blood vessels, and the mechanisms involved in this phenomenon are biologi-cally complex. Angiogenesis comprises short-term (minutes to hours) mechanisms such as vasodilatation and cell-cell adhesion; and long-term (hours or days) mechanisms involv-ing structural changes including migration and proliferation of endothelial cells, apoptosis or survival of vascular cells [76]. In the placenta, angiogenesis has been estimated by total capillary volume, surface and capillary length taking into account placental volume. These measures provide in-formation about overall growth, the net outcome of compet-ing processes (angiogenesis vs. vascular pruning and regres-sion), and are independent of the pattern of growth (i.e. branching vs. non-branching angiogenesis) [74].

The placenta microcirculation is a permeable and broadly selective vascular network [77, 78], and it constitute a unique human source of endothelial cells for functional studies in angiogenesis [79, 80]. There are few studies where human placental microvascular endothelial cells (hPMEC) have been characterized and used as a model of microvascualr endothelium [79-84]. However, it is known that hPMEC show a higher proliferative response to agonist (2-8 fold) compared with human umbilical vein endothelial cells (HUVEC) [79] or endothelium derived from chorionic ves-sels [80]. Unfortunately, there are not reports available re-garding hPMEC proliferations isolated from pre-eclamptic pregnancies [11, 37].

Evidences regarding functional markers of formation and function of vessels in the placenta in pre-eclampsia are con-tradictories [11, 37, 75]. A reduction in the total surface and length of the placental capillary [85] or no changes [75, 86] had been detected in pre-eclamptic placenta. Recent studies show that the placental capillary bed in pre-eclampsia does not affect oxygen diffusive conductance when compared with normal placentas [87]. Moreover, it has been demon-strated that intraplacental branching pattern of the umbilical artery is similar in placentas from pre-eclampsia and normal pregnancies [88]. However, other reports shown increased placental capillary branches formation and ramification in pre-eclampsia [74, 89]. In addition, other studies show in-creased CD34 [89, 90] or unaltered CD31 [91] (markers for endothelial cells) level in pre-eclampsia. Additionally, in transgenic mice expressing human angiotensin (i.e. hyper-tension model), CD31-positively immunostained placental microvessels at term were abnormal in size and number, and poorly supported by basement membrane compared with control animals [92]. Interestingly, a recent study shows that classification of pre-eclampsia in early (severe)- or late (mild)-onset might help to resolve some controversies re-garding vascular effects of the disease [12]. Thus, in a well-defined study groups and appropriate controls has revealed that late-onset pre-eclampsia affects minimally the placental villous and vascular morphology compared with gestational-age-matched controls. In contrast, early-onset pre-eclampsia was associated with reduced placental weight, volume of the intervillous space, terminal villous volumes and surface ar-eas of terminal villi [12]. These evidences suggest that in severe pre-eclampsia a reduction in placental angiogenesis could be responsible for the observed low blood flow; how-

ever, in mild pre-eclampsia it seems like there is no differ-ence or even increased placental angiogenesis [12, 89] main-taining a normal placental blood flow. The reasons explain-ing these differences in angiogenesis between mild vs. severe pre-eclampsia are unclear. However, it is interesting to speculate that the bioavailability of angiogenic factors, such as VEGF, sFlt-1, adenosine and NO in the fetal circulation in pre-eclampsia could alters microvascular endothelial cells proliferation and vessels formation in the human placenta [37, 93, 94].

ROLE OF ADENOSINE AND ADENOSINE RECEP-

TORS IN PRE-ECLAMPSIA AND ANGIOGENESIS

Adenosine and Pre-Eclampsia

Adenosine is a purinergic nucleoside derived from adenosine tri-phosphate (ATP), di-phosphate (ADP) and monophosphate (AMP) metabolism [95, 96],

or from S-

adenosine-homocysteine hydrolysis [97]. The biological ef-fects of adenosine, including angiogenesis, endothelial pro-liferation and permeability, and vasodilatation, among others [17, 18, 98] are associated with activation of adenosine membrane receptors [17]. At present, it has been identified four types of adenosine receptor i.e. A1, A2A, A2B and A3 [95]. Adenosine A1 and A2A receptors are known as high affinity receptors, whereas adenosine A2B and A3 receptors are referred as low affinity receptors [28, 99]. Adenosine A1 and A3 receptors are coupled to Gi/o protein and adenosine A2 receptors are coupled to Gs protein [95, 100-102]. High and low affinity adenosine receptors have been identified in the human placenta [103]. The use of several pharmacological approaches has allowed the characterization of A1 and A2 adenosine receptors in human and ewe placental vessels [66, 67]. More recently the mRNA for A2A, A2B and A3 adenosine receptors was identified in endothelium from human chori-onic vessels [68]

and A2A and A2B adenosine receptors

mRNA and protein were identified in primary cultures of hPMEC isolated from placentas from normal pregnancies and pre-eclampsia [84]. It has been reported that adenosine induces vasoconstriction via activation of A2B adenosine receptors and by thromboxane in this vascular bed in vitro, an effect avoided when endothelium was removed [68]. Moreover, high adenosine plasma level in the maternal and in the fetal circulation has been found in pre-eclampsia [19-26, 84]. Adenosine plasma level in the human umbilical vein blood was reported as 1.8 M, [19] a concentration that is significantly higher compared with adenosine level detected in normal pregnancies (~0.6 M) [104] Studies performed in primary cultures of hPMEC show a higher extracellular adenosine level (~4-fold) in cells isolated from pre-eclamptic compared with normal pregnancies [84]. Causes as well as consequences of an abnormally elevated extracellular adeno-sine level in pre-eclampsia are unclear; however, this phe-nomenon could be a potential adaptive mechanism, [37] which may be associated with vasodilatation or angiogenesis in pre-eclampsia, as characterized in other tissues such as heart, muscle or brain [105-107].

Adenosine and Angiogenesis

Adenosine acting through adenosine receptors stimulates endothelial cell proliferation and migration in the macro and

Placenta Angiogenesis in Pre-Eclampsia Current Vascular Pharmacology, 2009, Vol. 7, No. 4 479

microcirculation [27, 29, 30]. Adenosine could contribute up to 50-70% of the angiogenic response in some condition such as hypoxia [28] via a direct mitogenic effect on endo-thelium [108] or by regulating the production of pro-angio-genic substances, such as VEGF and interleukin 8 (IL-8) [18, 27, 109-113] or anti-angiogenic factors such as throm-bospondin 1 [96] from endothelial and immune cells [28,108]. It has been reported that activation of adenosine receptor with agonists such as NECA (5´-N-ethyl-carboxa-midoadenosine; a non-selective agonist), CGS-21680 (2-[p-(2-carbonyl-ethyl)-phenylethylamino]-5´-N-ethyl-carboxam-idoadenosine; A2A agonist) or DPMA (N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine; A2 agonist), increases VEGF mRNA level in bovine retinal en-dothelial cells [109]. The latter finding was blocked by CSC (8-(3-Chlorostyryl) caffeine; A2 antagonist) and mimicked by dibutyryl-cAMP suggesting that A2 adenosine receptors activation involves cAMP to increase VEGF expression in this cell type. Furthermore, in human retinal endothelial cells, NECA increased cell proliferation and the abundance of VEGF protein (2-16 fold), an effect blocked by anti-VEGF antibodies [110]. The signalling pathway associated to this phenomenon involves A2B adenosine receptor activa-tion and ERK1/2 phosphorylation [18]. In addition, these studies show that pharmacological A2B adenosine receptor blockage with IPDX (3-isobutyl-8-pyrrolidinoxanthine) or enprofylline prevented ERK1/2 phosphorylation and cell proliferation, migration and tube formation [18]. More re-cently, it has been reported that NECA increased intracellu-lar cAMP level due to activation of Gs proteins leading to ERK1/2 activation (i.e. phosphorylation) in HUVEC. NECA effect was associated with A2B adenosine receptor activation rather than other receptors since the selective A2B adenosine receptor antagonist MRS-1754 (N-(4-cyano-phenyl)-2-[4-(2,6-dioxo-1,3-dipropyl-2,3,4,5,6,7-hexahydro-1H-purin-8-yl)-phenoxy]acetamide) blocked NECA effect on ERK1/2 [30]. Interestingly, a reduced A2B adenosine receptor expres-sion induced by the use of a ribozyme selectively designed, blocked NECA effect on human retinal endothelium migra-tion and mouse endothelial proliferation [111]. Other studies show that NECA via A2B adenosine receptor activation in-creases the VEGF gene promoter activity, as well as VEGF mRNA level and protein abundance in the cultured medium in human microvascular endothelial cell line 1 (HMEC-1) [112]. It has also been shown that expression and protein abundance of VEGF is increased in HUVEC cultured in its physiological oxygen content (i.e. ~5% O2) exposed to NECA [27]. More recently, using primary cultures of human monocytes it has been shown that A1 adenosine receptor ac-tivation by the selective agonist CPA (N6-cyclo-pentyla-denosine) increased the VEGF level, an effect blocked with the selective antagonists WRC-0571 (N6-[endo-2'-(endo-5'-hydroxy)norbornyl]-8-(N-methylisopro-pylamino)-9-methyl-adenine) and CPX (8-cyclopentyl-1,3-dipropylxanthine) [113]. In addition, interaction between adenosine and VEGF had been also suggested in in vivo models where adenosine infusion (0.14 mg/kg/min, 6 h) increased (3-fold) the VEGF plasma level in humans [114]. Thus, adenosine could acti-vate adenosine receptor, probably A2A and/or A2B types, in-creasing cAMP intracellular levels and ERK1/2 phosphory-lation to induce VEGF expression and proliferation of hu-man microvascular endothelium. Unfortunately, nothing is

known regarding this potential mechanism(s) in human pla-cental microvasculature either in normal or pre-eclamptic pregnancies [26, 37].

VEGF SIGNALLING AND ANGIOGENESIS

Vascular endothelial growth factor is a family of growth factors including VEGF-A, B, C, D, and E, and PlGF [28, 115]. VEGF-A is the predominant isoform and has at least 5 splicing variants, i.e., VEGF-A121, A145, A165, A189 and A206

[115]. The predominant isoform VEGF-A165 (known as VEGF) is the only mitogen that acts specifically in endothe-lial cells [31, 33]. This factor is also considered as a survival factor, and due to its mitogenic effect promotes vascular ves-sels formation in both physiological and pathological condi-tions [32]. VEGF activate membrane receptors associated with tyrosine kinase activity, such as receptor type 1 (VEGFR1 or Flt-1), type 2 (VEGFR2, or KDR/Flk-1) and type 3 (VEGFR3) [35]. VEGFR2 activates several intracellu-lar signalling pathways including phospholipase C (PLC- ), PKC, NO and ERKs for DNA synthesis in endothelial cells [28, 35].

Role of Nitric Oxide in VEGF Signalling

Nitric oxide in synthesized by the enzyme family known as nitric oxide synthases (NOS) [102, 116, 117]. It is well established that NO could either activate soluble guanylate cyclase (sGC) increasing cGMP intracellular levels, or act directly on cysteine or tyrosine residues to induce nitrosyla-tion and nitration, respectively, leading to modulation of several cell functions [116-122]. Nitric oxide is part of the VEGF intracellular pathway in angiogenesis models and its effect can be proangiogenic [123, 124] or antiangiogenic [125, 126]. These seemingly contradictory biological effects may be explained by the action of different NOS isoforms and the different models used in experiments [127, 128]. Donors of NO, such as SNAP (S-nitroso-N-acetyl-penicillamine) or nitroglycerin, in a dose-depend manner increase DNA synthesis, an effect associated with prolifera-tion in rabbit coronary venular endothelial cells [127]. Stud-ies in endothelial NOS (eNOS) knockout animals (eNOS

-/-)

have reported a reduced vascular remodelling [129] and less collateral vessels formation [130] after ischemic injury com-pared with wild-type animals. Moreover, several evidences associate NO synthesis with proangiogenesis in human solid tumours [131-134] Interestingly, expression of inducible NOS (iNOS) isoform was associated with poor prognosis in neck cancer [133] or gastric cancer [135]. In addition, iNOS over-expression has been associated with increased invasion and angiogenesis in tumour models via stimulation of VEGF expression [132, 134, 136, 137]. However, the intracellular mechanisms for NO as inductor of endothelial cells prolif-eration are unclear. In HUVEC over-expression of sGC or incubation with the selective sGC agonist BAY 41-2272 ([5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyri-dine-3-yl]pyrimidin-4-ylamine]), increased cGMP intracellu-lar level and ERK1/2 phosphorylation, and stimulated cell proliferation and migration [138]. Nitric oxide has been also associated with bovine retinal endothelial cell proliferation via the formation of peroxynitrite (ONOO

-) [139]. Peroxyni-

trite induces VEGFR2 tyrosine autophosphorylation leading to activation of VEGF-mediated VEGFR2. Interestingly,

480 Current Vascular Pharmacology, 2009, Vol. 7, No. 4 Escudero et al.

FeTPPs [5,10,15,20-tetrakis (4-sulfonatophenyl) porphyri-nato iron (III)] a scavenger for ONOO

-, blocked VEGFR2

phosphorylation and endothelial cell proliferation induced by ONOO

- or VEGF [139], suggesting that VEGF could itself

induces ONOO- formation as a physiological mechanism to

regulate VEGFR2 phosphorylation. All together these stud-ies suggest that NO through cGMP and/or ONOO

- formation

could modulate endothelial cell proliferation induced by VEGF.

VEGF AND NO SIGNALLING IN PRE-ECLAMPSIA

In normal pregnancies, VEGF is crucial for trophoblast proliferation, development of embryonic vasculature, and growing of maternal and fetal vessels [140-142]. Thus, the importance of this growth factor and its receptors in normal gestation seems clear. However, in pre-eclampsia the expres-sion and activity of VEGF and VEGFR1 is contradictory. For example, high plasma levels of VEGF in umbilical vein blood samples, [143] as well increased mRNA level [141, 144]

and protein abundance [144, 145] in total placental ho-

mogenates had been reported. Other studies show low [146] or no change [147] in the levels of mRNA for VEGF and VEGFR1 in placenta homogenates from pre-eclampsia com-pared with normal gestations. Moreover, there are reports showing no differences in the level of VEGF of the human umbilical vein plasma [148], or even low levels of VEGF in the amniotic fluid in pre-eclampsia compared with normal pregnancies [149]. Certainly, the different methodologies used in these reports, as well as the source of samples, could explain the apparent controversy in the results. An interest-ing approach to clarify the role of VEGF as a proliferative factor is the use of a cellular model that allows the charac-terization of the VEGF synthesis mechanisms and regulation of its biological effects in the microcirculation of the pla-centa. Placental microvascular endothelium expresses VEGF [150] and functional VEGFR2, [83]

making these cells a

potentially adequate endothelial cell model for the study of VEGF expression and function.

THE SOLUBLE VEGF RECEPTOR TYPE 1 (SFLT-1)

sFlt-1 is synthesized in the cytotrophoblasts [93, 151] and has been associated with reduced angiogenesis in the mother [38, 152] and in cultured endothelial cells from the fetus [93, 153]. In pre-eclampsia, plasma sFlt-1 level is 5-fold higher in severe compared with mild pre-eclampsia [154-156]. Moreover, high sFlt-1 plasma level is considered as a predic-tive factor for severe pre-eclampsia with much more accu-racy than for mild pre-eclampsia [38, 39]. Interestingly, it has been reported that sFlt-1 level in the umbilical vein blood is 2-fold higher in pre-eclampsia compared with nor-mal pregnancies [157]. Unfortunately, the latter report did not indicate whether pre-eclampsia was mild or severe, but rather was mixed samples. Another study using the associa-tion between pre-eclampsia with or without IUGR in a small group of patients (8 patients per group) concluded that pre-eclampsia with IUGR was associated with higher level of sFlt-1 compared with the group of pre-eclampsia without IUGR, besides this differences were not statistically signifi-cant [158]. However, using this same stratification, Lask-owska and colleagues [159] in a small group of pregnant women showed high sFlt-1 level in umbilical plasma from

pre-eclamptic pregnancies associated with IUGR compared with pre-eclamptic pregnancies without IUGR [159]. Thus, it is feasible that severe pre-eclampsia is associated with a high sFlt-1 plasma level also in the fetal-placental circulation, which will decrease the bioavailability of VEGF reducing the signalling cascades to induce angiogenesis. Interestingly, a recent report suggests that the increased sFlt-1 plasma level detected in patients with pre-eclampsia is responsible of a reduced NO synthesis [160], suggesting this as a mechanism for the reduced angiogenesis detected in severe pre-eclampsia. Thus, ones could speculate that differences be-tween sFlt-1 plasma level in mild and severe pre-eclampsia could be responsible for the low angiogenic response in se-vere pre-eclampsia, but high or unaltered angiogenesis in mild pre-eclampsia [37].

CONCLUDING REMARKS

Pre-eclamptic pregnancies are associated with placental and fetal adaptation to an unfavourable maternal environ-ment. Endothelial cells proliferation and new vessels forma-tion could be a strategy by which the human placenta ensures the vascular blood flow toward the fetus. Thus, more likely the placental microvascular endothelial cells, hPMEC, are involved in this potential phenomenon of adaptation to the disease. An altered regulation in this adapative response is related with maternal disease severity. Thus, only severe rather than mild pre-eclamptic pregnancies showed low pla-cental vessel formation. This is a determinant difference, since short and long-term fetal complications inherent to pre-eclampsia are associated with severe rather than mild pre-eclampsia, and probably could be a feed-forward mechanism responsible for ischemic injury in the placenta during severe pre-eclamptic pregnancies. We certainly believe that a better understanding of the molecular mechanisms of these differ-ences will facilitate the suggestion of possible therapeutic protocols for the treatment of the pre-eclampsia. This review focused on unveiling cell signalling mechanisms triggered by the elevated extracellular adenosine concentration detected in human umbilical blood in pre-eclamptic pregnancies. This nucleoside in fact activates adenosine A2A/A2B receptors, and is involved in the regulation of VEGF expression and NO formation in the endothelium. We propose a modulation of VEGFR1/2 by VEGF in response to adenosine involving endothelial derived NO in the human placental microcircula-tion. This phenomenon could lead to a potential different angiogenesis dynamics in pre-eclampsia (see Fig. 1). We suggest that high plasma levels of sFlt-1 in severe pre-eclampsia could restrict placental angiogenesis explaining, at least in part, the proposed differential angiogenic response in severe and moderate pre-eclampsia.

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. C Escudero holds a MECESUP- and School of Medicine, Pontificia Universi-dad Católica de Chile- PhD fellowships (Chile). C Puebla holds a CONICYT PhD fellowship (Chile). F Westermeier holds a Pontificia Universidad Católica de Chile-PhD fel-lowship (Chile). We thank the researchers at the Cellular and

Placenta Angiogenesis in Pre-Eclampsia Current Vascular Pharmacology, 2009, Vol. 7, No. 4 481

Molecular Physiology Laboratory (CMPL) and Perinatology Research Laboratory (PRL) of the Pontificia Universidad Católica de Chile (PUC) for their contribution in the produc-tion 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