Overactive feto-placental nitric oxide system during pre- and perinatal insult

Department of Clinical and Experimental Medicine

Director Prof. Mario Petrini

Division of Obstetrics and Gynecology

University of Pisa

Thesis of the Residency Training Program in Obstetrics and Gynecology

OVERACTIVE FETO-PLACENTAL NITRIC OXIDE

SYSTEM DURING PRE- AND PERINATAL INSULT

Candidate:

Silvia PISANESCHI

Tutor:

Prof. Tommaso Simoncini

To: Chiara Greta Sofia Sveva

and Marco Filippo Iacopo Niccolò

" One of the most rewarding aspects in science is the inability to predict where a particular line of research may bring exactly. Indeed a wide-ranging research may end up to clarify an unresolved problem in a field that initially seemed very far".

INDEX

2.1.1 NO: a powerful modulator of vascular tone ……… 16

2.1.2 NO: general properties ……… 16

2.1.3 NO synthesis ………... 17

2.1.4 NO synthetase isoforms ………... 17

2.1.5 NO: mechanism of action ……… 18

2.1.6 Positive and negative regulators of no concentration in blood ………... 18

2.2. THE NO SYSTEM AND PREGNANCY…………..………. 19

2.2.1 NO and pregnancy……… 19

2.2.2 Involvement of NO on fertility and implantation ………... 20

2.2.3 NO is involved on the initial placentation mechanism counteracting recurrent miscarriage ……… 21 2.3 THE ROLE OF ENDOTHELIUM IN THE FETAL OXYGENATION ……….. 23

2.3.1 Endothelial cells and feto-placental haemodinamics ……….. 23

2.3.2 Normal fetal oxygenation ……… 23

2.4. FETO-PLACENTAL CHRONIC HYPOXEMIA LEADING TO FGR ………. 25

2.4.1 FGR: general features ………. 25

2.4.3 FGR and long-term morbidity ………. 26

2.4.4 Established etiological factors ………. 26

2.4.5 Emerging role of endothelium in the pathophysiology of FGR ……….. 26

2.4.6 Pathophysiology of the fetal response to chronic hypoxemia ………. 28

2.4.7 FGR and NO SYSTEM ………. 30

2.4.7.1 NO and FGR ………. 30

2.4.7.2 ADMA and FGR ………... 30

2.4.7.3 The role of l-Arginine ………... 31

2.4.8 Gene phenotype and endothelial function in FGR ……….. 31

2.4.8.1 Angiogenic factors in maternal and fetal blood ……… 31

2.4.8.2 extracellular matrix changes in feto-placental vessels ……….. 32

2.4.8.3 adhesion molecules expression in the placental bed ………. 33

2.5. PRE-NATAL METABOLIC RISK OF ADULT CARDIOVASCULAR DISEASES ………... 33

2.5.1 Prenatal programming and cardiovascular diseases later in life ………. 33

2.5.2 Fetal origins hypothesis ……….. 34

2.5.3 ADMA and CVD later in life ……….. 35

2.6. THE MECHANISM OF DELIVERY ………. 36

2.6.1 NO and labour ………. 36

2.6.2 Chemical and physiological parameters related to intra-partum hypoxia ………... 38

2.6.2.1 Fetal hypoxia – definitions ……… 38

2.6.2.2 Ph and base excess ……… 38

2.6.3 Feto-placental transitory hypoxemia during labor ……….. 40

2.6.3.1 Pathophysiology of the acute hypoxemia during labor……… 40 2.6.3.2 The physiological responses of fetus to reduced oxygen supply during normal,

uncomplicated labor (hypoxemia to moderate hypoxia) ……… 40

2.6.4 Development of severe hypoxia and asphyxia during labor ………... 42

Development of metabolic acidosis ……….. 43

Development of hypotension ……… 44

2.6.5 Responses to hypoxia during labor in fetuses afflicted by prenatal placental insufficiency .. 44

2.7 NO AND PERINATAL ADAPTATION ………. 46

2.7.1 Endothelium and pulmonary vascular tone at delivery ………... 46

2.7.2 The role of NO patway in the first week of life ……….. 46

2.7.3 Potential protective role of NO in the perinatal period ………. 47

3. WORKING HYPOTHESIS AND OBJECTIVES ……… 48

4. MATERIAL AND METHODS ………... 50

4.1. STUDY GROUPS ……… 50

4.1.1 First objective ……….. 50

4.1.2 Second objective ………. 53

4.2. DOPPLER VELOCIMETRIC ANALYSIS ……… 55

4.3. CELL CULTURES ……….. 55

4.4. NITRITE AND ENOS ACTIVITY ASSAY ………... 55

4.5. WESTERN IMMUNOBLOTTING ………. 56 4.6. NOHB ANALYSIS ……….. 56 4.7. ADMA ASSAY ………... 56 4.8. MINIARRAY ANALYSIS ……….. 56 4.9. STATISTICAL ANALYSIS ………... 59 5. RESULTS ………. 60 5.1. COMPENSATORY FETO-PLACENTAL UPREGULATION OF THE NO SYSTEM

DURING CHRONIC HYPOXEMIA LEADING TO FGR ……….. 60 5.1.1 NO2, NOHb and ADMA in umbilical blood ………... 60

5.1.2 eNOS function in HUVEC ……….. 63

5.1.3 Umbilical artery doppler analysis vs. NO markers in umbilical blood and cells from pregnancies with normal or restricted fetal growth ………

64 5.1.4 NO2 and NOHb in umbilical blood from twins with discordant fetal growth ………. 65 5.1.5 Blood NO2 and NOHb in newborns with normal or restricted growth ………... 66 5.1.6 Gene profile in HUVEC from fetuses with normal or restricted growth ……… 67 5.2. FETO-PLACENTAL NO SYSTEM DURING TRANSITORY HYPOXEMIA AT DELIVERY: PROGRESSIVE ACTIVATION WITH LABOR AND INTRA-PARTUM HYPOXIA

69 5.2.1 Concentration of NO2, NOHb and ADMA in umbilical blood: effect of labor ……….. 69 5.2.2 eNOS function in HUVEC: effect of labor ………. 70 5.2.3 Concentration of NO2, NOHb and ADMA in umbilical blood: effect of intra-partum hypoxia ………...

71 5.2.4 eNOS function in HUVEC: effect of intra-partum hypoxia ………... 72 5.2.5 Concentration of NO2, NOHb and ADMA in umbilical blood: effect of intra-partum hypoxia depending on the mode of delivery ………..

73 5.2.6 eNOS function in HUVEC from pregnancies with intra-partum hypoxia: effect of labor …. 74 5.2.7 Blood NO2and NOHb in newborns from different modes of delivery ………... 75

6. DISCUSSION ………... 76

6.1. SUMMARY OF THE FINDINGS ……….. 76

6.2. OVERACTIVE FETO-PLACENTAL NO SYSTEM DURING CHRONIC HYPOXEMIA AND FGR ………..

77 6.3. POTENTIAL “SIGNATURE” FOR FGR ENDOTHELIUM IN THE FETO-PLACENTAL CIRCULATION THROUGH A MECHANISM OF EPIGENETIC PROGRAMMING ……….

80

6.5. COMPENSATORY ROLE OF THE FETO-PLACENTAL NO SYSTEM DURING TRANSITORY HYPOXEMIA OF LABOR………..

82 6.6. INVOLVEMENT OF NO ON THE BEGINNING OF NEONATAL BREATHING … 83 6.7 PROGRESSIVE ACTIVATION OF THE FETO-PLACENTAL NO SYSTEM COUNTERACTING THE INTRA-PARTUM HYPOXIA ………...

84 6.8 THE NO SYSTEM AND THE RESPONSES OF THE VULNERABLE FETUS TO INTRAPARTUM HYPOXIA ………

85

7. CONCLUSION ………. 86

ABBREVIATIONS

ACE1: Angiotensin I converting enzyme (peptidyl-dipeptidase A) 1, ACE2: Angiotensin I converting

enzyme (peptidyl-dipeptidase A) 2, ACTH: adenocorticotropic hormone, ADMA: asymmetric dimethylarginine, AGA: appropriate for gestational age, AGTR2: Angiotensin II receptor, type 2, Akt: protein chinasi B, alCS: cesarean section after labor, ANXA5: Annexin A5, bFGF: basic fibroblast growth factor, BE: base excess, BW: bodyweight, BH4: tetrahydrobiopterin, Ca: calcium ion, CaCl2: chlorure de

calcium, CaM: calmodulin, CDH5: Cadherin 5, Cys: cisteine, CO: carbon monoxide, COL18A: collagen type XVIII alpha 1, COX-2: cyclo-oxygenase-2, CT: control twin, CTG: cardiotocography, CVD: cardiovascular diseases, CXCR5 or BLR1: chemokine (C-X-C motif) receptor 5, DDAH: dimethylarginine dimethylaminohydrolase, DMEM: Dulbecco’s Modified Eagle Medium, ECGF1 or TYMP: endothelial cells growth factor1 or thymidine phosphorylase, eCS: elective cesarean section, ECM: extracellular matrix,

EDHF: endothelium-derived hyperpolarizing factor, EDTA: ethylenediaminetetraacetic acid, eNOS or

NOS3(Arroyo, Anthony et al.): endothelial constitutive NOS, EPR: electron paramagnetic resonance, ESR: electron spin resonance, ET: endotelin, FASLG: fas ligand member 6, FBS: fetal bovine serum, Fe: iron ion, FGR: fetal growth restriction, FHR: fetal heart rate, cGMP: guanosine 3-5 cyclic monophosphate, Gly: glycine, HELLP syndrome: hemolysis, elevated liver enzymes, low platelets, HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulforic acid), HES: endometrial surface epithelial cells, HIE: hypoxic-ischemic encephalopathy, HIF-1: hypoxia inducible factor 1, HO-1: inducible heme oxygenase-1, HPA: hypothalamic-pituitary-adrenal, HPG: hypothalamic-pituitary-gonadal, HUVEC: human umbilical vein endothelial cells, Hyp: intra-partum hypoxia, ICAM-1 or CD54: intercellular cell adhesion molecule-1,

IFN-γ: interferon-γ, 1: interleukin-1, 1β: interleukin-1β, 4: interleukin-4, 6: interleukin-6,

IL-8: interleukin-8, IL-10: interleukin-10, iNOS orNOS2 (Arroyo, Anthony et al.) or NOS 2A: inducible

NOS, ITGB1: integrin beta 1, FGR: Fetal Growth Restriction, KDR: kinase domain receptor, L: length,

LDL: low density lipoprotein, L-NMMA: NG-monomethyl- L-arginine, LPS: lipopolisaccaride, LTs:

leukotrienes, mRNA: messenger ribonucleic acid, M: mean, MAPK: mitogen-activated protein kinases,

metalloproteinase 13 [human collagenase], MMP-2: metalloproteinase 2 [gelatinase A], MMP-3: metalloproteinase 3 [stromelysin-1], MMP-8: metalloproteinase 8 [neutrophil collagenase], MMP-9: metalloproteinase 9 [gelatinase B], MMPs: matrix-metalloproteinases, MT-MMPs: membrane-type matrixmetalloproteinases, NF-kB: nuclear factor-kB, nNOS or NOS1 [type I]: neuronalconstitutive NOS,

NO: nitric oxide, NOS: nitric oxide synthetase, Nox: normoxia, NOx: nitrogen oxides, •NO2: nitrogen

dioxide, NOX: normoxia, O2: oxygen, O2-: superoxide anion, [•OH]: oxidril radical, [ONOO-]:

peroxinitrite, PCR: polymerase chain reaction, PInd: P index, PDGF: platelet-derived growth factor, PE: pre-eclampsia, PECAM-1 or CD31: platelet-endothelial cell adhesion molecule-1, PF4: platelet factor 4,

PGHS-2: prostaglandin endoperoxide synthase-2, PGs: prostaglandins, PI: pulsatility index, PlGF:

placental growth factor, PT: prior to term, PTGS2: prostaglandin endoperoxide synthase-2, RAAS: renin-angiotensin aldosterone system, RBC: red blood cells, RNA: ribonucleic acid, s-Eng: soluble endoglin, SD: Std. Deviation, S/D: systo/dyastolic rate, SDMA: symmetric dimethylarginine, SDS-PAGE: sodium dodecyl sulphate-polyacrylamide gel electrophoresis, SE: Std. Error, SELL: Selectin L, sFlt-1: soluble fms-like tyrosine kinase 1, SGA: small for gestational age, SHQ: super higher Q, NOHb: S-nitrosohemoglobin, ST: small twin, s-TM: soluble thrombomodulin, sVEGFR-1: soluble VEGF receptor-1,

T: term, TGF-β: transforming growth factor-β, THBS1: thrombospondin-1 gene, TIMP1: tissue inhibitors

of metalloproteinase 1, TIMPs: tissue inhibitors of metalloproteinases, TNFSF10: tumor necrosis factor 10,

TNF-α: tumor necrosis factor-α, TSP1: thrombospondin-1 protein, Tx: thromboxane, TxA2: thromboxane

A2, US: ultrasound, VCAM-1 or CD106 : vascular cell adhesion molecule-1, VD: vaginal delivery, VEGF: vascular endothelial growth factor, vWF: von Willebrand factor.

Key Words: nitric oxide, nitroso-hemoglobin, asymmetric dimethylarginine, vascular endometrium,

ABSTRACT

Background

Endothelium-derived nitric oxide (NO) is a highly reactive inorganic free radical with widespread biological actions, including vascular regulation, neurotransmission, hormone secretion and inflammation.

Potential roles for NO in the human uterus include vasodilatation (both before implantation, and in the uteroplacental and systemic circulation during pregnancy).

Nitric oxide may also be involved in diseases of pregnancy, from unexplained infertility and recurrent miscarriage and/or defective placentation in the first period of gestation. During the third trimester of pregnancy, a change in the NO production may be involved in pre-eclampsia and fetal growth restriction (FGR). NO may also have a compensatory function, and several situations in the adult document this possibility.

This thesis aimed to verify whether the NO activation represents a unifying mechanism for the preservation of blood delivery to the fetus during different adverse conditions.

The working hypothesis was that endothelial cells from feto-placental vessels are key determinants of any situation in which the fetus is exposed to a hypoxic insult and requires an adequate amount of oxygen for well-being, including chronic hypoxemia leading to FGR and transitory hypoxiemia during normal or disturbed labor.

Further we aimed to define the nature of any linkage between NO and FGR and, coincidentally, provide a possible insight into the alleged negative impact of FGR on adult health.

Methods

The approach was comprehensive and included: (i) measurement of NO and its main metabolite, nitrite (NO2), along with the natural NO synthase inhibitor asymmetric dimethylarginine (ADMA); (ii) analysis of Doppler velocimetry in umbilical arteries; and (iii) assessment of gene profile in umbilical vein endothelial cells (HUVEC) collected at the time of delivery.

Study comprised term pregnancies with average (n =40) or small-for-gestational age body weight (n = 20) (both scheduled for umbilical Doppler velocimetry at 36 wk), pregnancies with isolated preterm FGR (n = 15) and bi-chorial, bi-amniotic twin pregnancies with discordant fetal growth (n = 12).

Cord blood (artery and vein) was collected in all cases, while peripheral blood (heel sampling) was obtained from certain newborns at the time of delivery and 24 or 72 h afterwards.

A second analysis was performed on the sub-group of pregnancies where delivery of a normal newborn had occurred at term, either vaginally or through cesarean section without/with prior labor (n = 20). In addition, cases from the same cohort but not considered earlier, where delivery had been complicated by fetal hypoxia (n = 15), were examined. Then, collective values were cross-analyzed depending on the mode of delivery and the presence/absence of fetal hypoxia. Separately, the same variables were measured over the first three days of life in term neonates from vaginal and cesarean (with/without labor) delivery uncomplicated by hypoxia.

Results

The NO system within the placenta and the fetus itself may be important in maintaining a suitable oxygenation of the offspring through any intervening hypoxic insult.

Compensatory feto-placental up-regulation of the NO system during FGR was shown. In detail, umbilical blood nitrite (p < 0.001) and S-nitrosohemoglobin (p = 0.02) rose with fetal growth restriction while asymmetric dimethylarginine decreased (p = 0.003). Nitrite rise coincided with an abnormal Doppler profile from umbilical arteries. Our analysis of the expression of a wide set of endothelial genes suggests that this phenomenon is part of a re-setting of endothelial function, as an adaptative event to sustain placental blood flow. In fact, in the presence of fetal growth restriction, umbilical vein endothelial cells produced more nitrite and also exhibited reciprocal changes in vasodilator (upwards) and vasoconstrictor (downwards) transcripts.

Moreover, elevation in blood nitrite and S-nitrosohemoglobin persisted post-natally in the fetal growth restriction offspring, potentially modifying the endothelial phenotype and possibly representing an element of risk for cardiovascular disease in adult life.

Similarly to FGR, where NO may be of use to counteract chronic impairment in oxygen inflow to the fetus, fetal and placental NO may also be important to facilitate blood flow to the fetus during labor and delivery.

In particular, active labor was associated with higher NO and NOHb concentrations in the umbilical vein blood. Accordingly, HUVEC from labor-based deliveries presented greater eNOS expression and activity. The same blood variables, however, presented an opposite trend in the umbilical artery. Further activation of the NO system occurred with deliveries complicated by offspring hypoxia along with a fall of ADMA levels. This set of responses may help adapt to post-natal breathing, extending the potential protective role of NO to the peri-partum period; in fact, the upward change in NO activity progressed over the first 24 hrs after birth to subside by 72 hrs.

Conclusions

The role of NO in diverse uterine conditions may have great clinical implications in developing therapeutic strategies to prevent NO-related disorders. Indeed, if a role for NO is confirmed, pharmacological modification of NO activity may lead to novel therapeutic applications. Moreover, the NO system within the placenta and the fetus itself may be important in maintaining a suitable oxygenation of the offspring through any intervening hypoxic insult. FGR is typified by increased nitric oxide production during pregnancy and after birth. This response is viewed as an adaptive event to sustain placental blood flow. However, the phenotypic characteristics of endothelial cells linked to the synthesis of NO might contribute some sort of imprinting to the vulnerable newborn determining functional vascular changes that may be important for postnatal adaptation but that may as well be long-lasting, possibly programming the infant in the long-term. As the NO system is a key player in preserving fetal oxygen availability in chronic conditions of increased demand, transitory hypoxemia during delivery is associated with enhanced NO function in the feto-placental district with a concomitant greater utilization of the agent by the fetus. This normal event is magnified with intra-partum hypoxia and, in all cases, persists in the immediate post-natal period. We regard this set of

changes as a protective mechanism whose finality is to maintain an adequate oxygenation of the fetus through delivery with an attendant smooth transition from intra- to extra-uterine life.

1. PREMISE

Endothelium- derived nitric oxide (NO) is a highly reactive inorganic free radical with widespread biological processes, including vascular regulation, neurotransmission, hormone secretion and inflammation, and plays a crucial role in the reproductive system.

Throughout pregnancy, NO is useful in maintaining a low resistance in the placental circulation, hence the conditions for an adequate exchange of gases and nutrients between mother and fetus. Situations where feto-placental exchanges are impaired, as it happens with acute or chronic hypoxia, may affect the fetus in various ways in any state of the development process.

With this premise, it was of interest to examine whether instances, where fetal oxygenation is chronically or acutely disrupted, are accompanied by enhanced NO production by the feto-placental unit.

Exemplary in this context is the fetal growth restriction (FGR), often associated with a chronic feto-placental vascular dysfunction conceivably involving endothelial cells.

Additionally, the labor-based delivery, where uterine contractions interfere with placental perfusion, is likely to cause acute disruptions of fetal gas exchanges.

Here, we describe a significant activation of the NO system in the feto-placental circulation during these events, when the fetus requires a compensatory oxygen support for well-being.

2. BACKGROUND

2.1. THE NO SYSTEM

2.1.1 NO: a powerful modulator of vascular tone

Nitric oxide (NO) is considered as the most powerful vasodilator secreted by endothelial cells (Ignarro 1990). Many studies have revealed the importance of NO as a major mediator in numerous biological processes in humans. This ubiquitous molecule is considered to be an important paracrine messenger controlling changes in several physiological and pathophysiological events that occur in endocrine organs and to be able to prevent endothelial cell dysfunction and vascular degenerative processes. Deranged NO synthesis by endothelial cells is associated with decreased vascular dilatation, hypertension and enhanced atherosclerosis (Hansson 2005).

NO can be produced in macrophages, lymphocytes and neutrophils (Moncada 1992), and is an important determinant of immune and inflammatory response. The bactericidal, fungicidal, viricidal and tumoricidal activities of macrophages are determined in part by generation of NO, hence its levels have been found to be increased with infections. In addition, it seems that NO may act as a promoter of growth (Sammut, Foresti et al. 1998). Several evidences have also indicated the neuromodulator NO action (Ignarro 1990). NO has been shown to be active in a number of vascular beds including brain, lung, and kidney (Myatt, Brewer et al. 1991) (Swain, Le et al. 1997) (Vaziri, Ni et al. 1998). NO is also secreted by the placenta (Zarlingo, Eis et al. 1997) and has been shown to modulate feto-placental and utero-placental blood flow (see afterward). It seems that NO release is related with an increased shear-stress of endothelial cells.

2.1.2 NO: general properties

NO is an uncolored gas and a free radical, that permeates quickly the biological membranes (Ignarro 1990). It reacts quickly with water and molecular oxygen, converting into nitrite and nitrate, which are its stable by-products (Stuehr and Griffith 1992). It can also combine with the superoxide anion (O2-), forming peroxinitrite [ONOO-] (Ignarro 1990), a powerful oxidant, that, according to circumstances, can work as "scavenging system" of oxidril radical [•OH] and nitrogen dioxide (•NO2) (Ischiropoulos, Zhu et al. 1992). Moreover, NO may react with ozone, yielding in activated •NO2, with oxyhemoglobin, producing

methemoglobin and nitrate, with transition metals. Finally, it has been demonstrated that NO reacts with a variety of molecules including heme-containing enzymes such thiols to form S-nitrosothiols (Lyall, Greer et al. 1996).

2.1.3 NO synthesis

NO is synthesizedby a family of three isoforms of NOS: neuronalconstitutive (nNOS or NOS1 [type I]), inducible NOS (iNOS orNOS2 [type II]), and endothelial constitutive (eNOS or NOS3[type III]), from a guanidine nitrogen of L-arginine, which is transformed in L-citrulline (Moncada, Palmer et al. 1991).

2.1.4 NOsynthetase isoforms

All isoforms bind Calmodulin (CaM) and catalyze the conversion of L-arginine to L-citrulline, resulting in NO synthesis and vasorelaxation (Moncada, Palmer et al. 1991). The nNOS reversibly binds CaM, only in presence of elevated levels of Ca intracellular. The iNOS is found in nucleate cells of mammals, including macrophages, neutrophils, smooth muscle, fibroblasts, endothelial cells (Gross, Jaffe et al. 1991). It can be induced by products from gram-negatives (as lipopolisaccaride, LPS) or gram-positive bacteria or by the cytokines, such as interleukin-1 (IL-1), tumor necrosis factor (TNF-α) and interferon-γ (IFN-γ). Often, the induction of NOS requires a synergism between two or more cytokines and/or bacterial products (Gross, Jaffe et al. 1991). This NOS is defined Ca-independent, because it does not increase its activity in response to increases of intracellular Ca. However, it requires physiological concentrations of Ca typical of the cells at rest. The NOS III, although it has been identified for the first time in endothelial cells, is present in various cellular types, among which endothelium (Gross, Jaffe et al. 1991). It has been ascertained that the eNOS binds to specific membrane’s sites, named caveolae. Caveolae are intussusceptions of 50-100 nm of the plasma membrane, present in endothelium, in skeletal and cardiac muscle and other tissues, but absent in neurons, composed mainly of glicosphyngolipids, cholesterol and of structural proteins named caveolines (Michel, Feron et al. 1997). The association of NOS III to cellular membrane is regulated from two modifications with fatty acids: a co-translation N-miristhoylation in glycine (Gly) and a post-translation palmithoylation in cysteine (Cys). The miristhoylation is irreversible, while palmithoylation is reversible

(Michel, Feron et al. 1997). NOS III is inactive when bound to caveolae, the increase of Ca and the association with the complex Ca/CaM favors the separation of eNOS from caveolin and its activation; a reduction of Ca favors the inverse process (Michel, Feron et al. 1997). NOS is present in human pregnant myometrium, placental villous trophoblast, and fetal membranes (Ledingham, Thomson et al. 2000), and iNOS has been immunolocalized to human myocytes.

2.1.5 NO: mechanism of action

The vasorelaxant action of NO occurs through the stimulation of guanylcyclase in nearby smooth muscle cells, with a consequent increase of guanosine 3-5 cyclic monophosphate (cGMP) (Moncada, Palmer et al. 1991). The mechanism by means of which the cGMP increase contributes to NO effect is not completely clear, but it seems to involve myosin light chain phosphorylation. Alternatively, it can be related to the block of Ca influx in smooth muscle cells (Lockette, McCurdy et al. 1989). Moreover, it seems that NO binds with elevated affinity to Fe of hemoprotein heme, creating a nitroso-heme adduct, which is relatively stable (Ignarro 1990). The link with hemoglobin’s heme impedes NO interaction with cytosolic guanylcyclase, potentially explaining the hemoglobin inhibiting action on NO vasodilator effect ("scavenger action").

2.1.6 Positive and negative regulators of NO concentration in blood

In vivo changes of NO bioavailability can derive from reduced synthesis from dysfunctional endothelial cells, but also because of increased endogenous inhibitors of NOS. In 1992, McCall and co-workers were the first to describe substances that show structural homology to L-arginine, but differ from it in that they contain one or two methyl groups (McCall and Vallance 1992). These analogous of the arginine, among which monomethyl- L-arginine [L-NMMA], symmetric dimethylarginine [SDMA] and ADMA, act as competitive inhibitors of NOS, with varying affinity for the three isoforms (Moncada, Palmer et al. 1991). There is evidence that these analogues inhibit NO production in a concentration-dependent manner. The ratio of the concentrations of L-arginine and ADMA determines the activity of NOS in vivo and, thereby, influences vascular function (MacAllister, Fickling et al. 1994). However, even these negative regulators operate under a control. For instance, ADMA is degraded to dimethylamine and citrulline by two isoforms

of dimethylarginine dimethylaminihydrolase (DDAH) (MacAllister, Parry et al. 1996). Moreover, hormones, growth factors and cytokines may inhibit iNOS. They include glucocorticoids, transforming growth factor-β [TGF-β] -1, -2, -3, interleukin-4 (IL-4), interleukin-10 (IL-10), macrophage deactivation factor [MDF] (Nathan 1992), interleukin-8 (IL-8), platelet-derived growth factor [PDGF] (McCall and Vallance 1992). In addition, the relevance for dysfunctional vascular control of altered circulating amounts of NO-protein conjugates that act as carriers of NO in the bloodstream, such as S-NOHb (Singel and Stamler 2005), is increasingly recognised. These products may act as a store for NO (Stamler, Simon et al. 1992). The role of the formation of nitrosothiols in normal pregnancy and FGR is not known.

2.2. THE NO SYSTEM IN EARLY PREGNANCY

Human studies and animal models indicate that NO has a key role in several reproductive phenomena, including pregnancy. Conditions such as recurrent miscarriage, deranged implantation and placentation and some complications of the third trimester of pregnancy, including pre-eclampsia and intrauterine growth restriction, have been associated with abnormal function of the nitric oxide system in the feto-placental circulation.

Regulation of NO synthesis in endothelial cells is a primary target of sex steroid hormones. Synthesis of estrogens and progesterone increases dramatically throughout pregnancy mostly due to fetal and placental production. During pregnancy elevations of 17beta-estradiol, estrone, estriol are found due to placental endocrine activity. Pregnancy-specific estrogens can also be found, such as estetrol, which rapidly disappears upon delivery. Estrogens stimulate the synthesis of NO in endothelial cells through the enhancement of eNOS gene expression (Kleinert, Wallerath et al. 1998) as well as through non-transcriptional activation of eNOS enzymatic activity (Simoncini, Hafezi-Moghadam et al. 2000) (Haynes, Sinha et al. 2000) (Hisamoto, Ohmichi et al. 2001) (Chen, Yuhanna et al. 1999) (Kim, Lee et al. 1999). Rapid intracellular events such as activation of tyrosine kinases, mitogen-activated protein kinases and Akt/protein kinase B mediate these latter actions, leading to phosphorylation on (Ser1177)-eNOS and to its consequent activation (Simoncini, Hafezi-Moghadam et al. 2000) (Hisamoto, Ohmichi et al. 2001)

(Simoncini and Genazzani 2003). Estrogen modulation of eNOS has been widely studied and represents one of the foremost vascular actions of these steroids, overall resulting in vascular protective effects

(Chambliss and Shaul 2002) (Simoncini and Genazzani 2003).

2.2.2 Involvement of NO on fertility and implantation

In the past 10 years, NO has established itself able to play a decisive role in regulating multiple functions within the female (ovary, oviduct, cervix and vagina, placenta) as well as the male (regulation of penile erection, testis, sperm motility and fertilization, sexual behaviour) reproductive system (Rosselli, Keller et al. 1998).

Recent findings provide evidence that NO, both ovarian cell-delived and vascular endothelial cell-derived, plays an important role in the physiology and biology of the ovary with regard to regulation of folliculogenesis and ovulation (Rosselli, Keller et al. 1998), suggesting this metabolite as a vital molecule controlling hypothalamic-pituitary-gonadal (HPG) axis (Dixit and Parvizi 2001).

Implantation of the embryo is a critical event in pregnancy. In humans, peri-implantation pregnancy loss may contribute to more than 20% of unexplained infertility. Approximately one-third of human pregnancies end in spontaneous abortions, two-thirds of them occurring prior to the clinical detection of pregnancy (Wilcox, Weinberg et al. 1988). Successful implantation of blastocysts is strictly dependent on the synchronic interaction between cells of maternal endometrium and conceptus, resulting in a cascade of cellular and molecular mechanisms. These events occur in the endometrium during the peri-implantation period of the fertile cycle, and induce an adequate blood supply to the implantation sites (Beier, Hegele-Hartung et al. 1994).

During early pregnancy, the surrounding endometrial stroma undergoes a dramatic differentiation into the decidua, a specialized, well-vascularized tissue that encapsulates the developing embryo. Decidual cells are believed to play a key role in providing nutrients to the embryo, and in controlling trophoblast invasion (Parr and Parr 1985). Additionally, the decidual cell reaction occurs in response to either blastocysts or artificial stimuli and is always preceded by an increase in endometrial vascular permeability which is normally initiated at the antimesometrial sites where blastocysts implant (Psychoyos 1973). Finally, cytotrophoblast

invasion, encompassing dynamic changes in cell–cell and cellmatrix interactions, can be viewed as an inflammatory reaction (Chwalisz, Winterhager et al. 1999).

In a human pilot study, it has also been observed that NO production in embryos is positively associated with the growth potential of a developing conceptus (Battaglia, Ciotti et al. 2003).

Since NO acts as a local mediator of inflammatory response and directly regulates the activity of matrix metalloproteinases (MMPs) (Trachtman, Futterweit et al. 1996), it is likely that the continuous generation of NO into the lumen seems to facilitate the process of implantation via modulation of anchoring proteins (Rosselli, Keller et al. 1998), throughout the stimulation of the MMPs production (Tamura, Nakanishi et al. 1996).

Additionally, NO might also contribute to the vasodilation of the maternal systemic circulation at the implantation site and it in turn suggestively aids in trophoblast penetration into spiral arterioles (Ahmed, Dunk et al. 1997).

The presence of a large amount of iNOS at the feto–maternal interface throughout the first trimester of pregnancy suggests that iNOS may be an important factor involved in implantation (Norman and Cameron 1996).

These findings suggest that NOS may represent a new target for novel therapeutic agents capable of promoting or inhibiting implantation.

Up-regulating uterine NO production with either the NO substrate L-arginine and NO donors alone or in combination with progesterone might have beneficial effects on pregnancy outcome during assisted conception.

The effects of NOS inhibitors in combination with antiprogestins point to a novel method for controlling fertility, particularly by enhancing the efficacy of antiprogestins used for endometrial contraception, menstrual induction and post-coital contraception.

2.2.3 NO is involved on the initial placentation mechanism counteracting recurrent miscarriage

It is possible that NO produced in large quantities in the presence of NOS plays a paracrine role in regulating uterine blood flow and immunosuppression for successful pregnancy.

In fact, recent evidences suggest that progesterone, potentiating the NOS expression in endometrial cells, also stimulates the production of progesterone-induced blocking factor (PIBF), which in turn induces a Th2 response and down-regulates NK activity and exerts an anti-abortive effect (Norwitz, Schust et al. 2001) (Szekeres-Bartho and Wegmann 1996).

Additionally, authors reported that homozygosity for a common 677C3T mutation in the 5,10- methylenetetrahydrofolate reductase (MTHFR) gene, which is associated with afterload homocysteine concentrations, leads to a twofold to threefold higher risk of recurrent early pregnancy loss. The 5-MTHFR seems to maintain in the reduced form the tetrahydrobiopterin (BH4) and to simulate three-dimensionally the BH4, directly activating eNOS (Nelen, Blom et al. 2000). This may be confirm the protective role of NO in the pregnancy preservation.

Furthermore, numerous papers highlighted that NO modulates prostaglandin production (Mollace, Muscoli et al. 2005). The importance of Prostaglandin E2 in early pregnancy has been shown, exerting its inhibitory effect on T cell activation and proliferation and favouring the synthesis of matrix metalloproteinases, which assist cytotrophoblast invasion of the decidua (Corcoran, Kibbey et al. 1995).

Finally, a large amount of NOS at the feto–maternal interface throughout the first trimester of pregnancy suggests that NOS may be an important factor involved in the mechanism of effective placentation.

In detail, Martin & Conrad (Martin and Conrad 2000) observed a discernible degree of transcripts for eNOS in extravillous trophobalst cells in human first trimenster placenta samples, while a large amount of iNOS protein is strongly expressed in human syncytiotrophoblastic cells in the first trimester (Yoshiki, Kubota et al. 2000). Instead, nNOS has been localized in trophoblast cells from human term placenta (Sanyal, Nag et al. 2000), but not in first trimester placental samples (Al-Hijji, Andolf et al. 2003).

Novel therapeutic agents capable of promoting effective placentation, as an up-regulating uterine NO production with either the NO substrate L-arginine and NO donors alone or in combination with progesterone, might have also implications for the management of early pregnancy disorders, including recurrent abortions.

2.3

THE

ROLE

OF

ENDOTHELIUM

IN

THE

FETAL

OXYGENATION

The feto-placental vascular tone control depends on a set of molecular factors (Vane, Anggard et al. 1990). Endothelial function is the greatest determinant for feto-placental vasodilatation and vasoconstriction (Vane, Anggard et al. 1990). Endothelial cells are primarily involved in the vascular tone control (Trochu, Bouhour et al. 2000), acting through fast and not genomic processes which generally control the endothelium-dependent vasodilatation (Simoncini, Genazzani et al. 2002, Simoncini and Genazzani 2003, Simoncini, Fornari et al. 2005). In particular, these cells are able to adapt themselves to fast environmental changes, through the synthesis and release of numerous biologically active molecules, thus conferring to the vessels the capacity of modulating blood flow in relation to the tissue metabolic request (Trochu, Bouhour et al. 2000). On the other hand, under persistent environmental changes, endothelial cells are subject to middlelong term changes of their functional phenotype through the induction or inhibition of the gene expression that are significant for endothelial function and dysfunction (Simoncini, De Caterina et al. 1999, Simoncini and Genazzani 2000, Libby 2003), in order to adapt to particular functions (Libby 2003).

Regulatory factors released from endothelial cells are critical for the control of feto-placental hemodynamics, ensuring an adequate placental blood flow and fetal oxygenation (Rutherford, McCarthy et al. 1995).

Increased feto-placental tone and reactivity seems to be due, in part, to alterations in vasoactive mediators such as endothelin (ET), prostacyclin, thromboxane (Tx), and NO. In fact, it has been demonstrated that the endothelial cells constitute the largest source of the ET which, acting through two receptor types, may produce either constriction or relaxation of vascular smooth muscle.

2.3.2 Normal fetal oxygenation

In uterine life, the oxygen tension and drive for effective fetal gas exchange is quite different from the adult population. Sir Joseph Barcroft summarized this in 1946 with the phrase “Mount Everest in utero”, comparing the partial pressure of oxygen in the fetus with what we find in humans at the top of Mount

Everest (Bennet and Gunn 2009). However, this does not mean that the fetus spends nine months in lack of adequate oxygenation. Normal fetal physiology involves several adaptive mechanisms, many similar to those known from acclimatization to high altitude. These adaptive mechanisms permit the fetus to achieve a level of oxygen consumption similar to extra uterine life, in fact exceeding its needs under normal conditions.

An important mechanism for sustaining adequate fetal oxygenation is the maintenance of high blood flow rates in fetal tissue. This is a result of the high fetal cardiac output, mainly due to the rapid fetal heart rate. An optimized gas exchange across a large respiratory surface, i.e. the placenta, is also significant, as well as improved oxygen transport by hematologic adaptations. Such hematologic adaptations include high fetal hemoglobin concentrations, increasing oxygen binding capacity, and a shift of the hemoglobin dissociation curve to the left (as opposed to the right-ward shift seen in adults exposed to high altitudes), resulting in enhanced oxygen affinity in the blood. The combined increase in both capacity and affinity provides the fetus with high blood oxygen stores, something that may defer anaerobiosis in periods of reduced oxygen supply. To some degree this will also compensate for the slight impaired tissue oxygenation resulting from the shift of the hemoglobin dissociation curve.

In order to ensure sufficient oxygenation of vital fetal organs, there are also metabolic adjustments in the tissues leading to reduced oxygen demands. The metabolic rate of the immature fetal brain is particularly low compared to adult tissues. This is partly due to the lower cell membrane permeability resulting in delayed depolarization, but is also caused by reduced release of excitatory amino acids from nerve terminals (Singer 1999).

Another significant contribution to adequate fetal oxygenation in utero, as well as after birth, is the precise and complex regulation of fetal heart rate (FHR). Chemoreceptors in the aorta and the carotid artery monitor changes in the circulating concentration of oxygen, carbon dioxide, and hydrogen ions. Any changes in arterial pressure are registered by baroreceptors in several of the large systemic arteries in thorax and the neck, the aortic arch and the carotid artery in particular. Together with medullary cardiorespiratory centers, these receptors feed the autonomic nervous system with information about the body’s circulatory and respiratory state, helping to adjust the FHR to the body’s need.

2.4. FETO-PLACENTAL CHRONIC HYPOXEMIA LEADING TO FGR

FGR affects up to 8% of all pregnancies (Brar and Rutherford 1988) (Pollack and Divon 1992). Small for gestational age (SGA) is defined as less than 10 percent of predicted fetal weight in relation to growth curve (Clinical Green Top Guidelines 2002. The investigation and management of the Small-for-Gestational-Age Fetus. Royal College of Obstetricians and Gynecologists). FGR refers to an estimated fetal weight below the 5th centile for gestational age or postnatal birth weight below the 3rd centile (Schiessl, Mylonas et al. 2005). The modern ultrasound (US) techniques for prenatal diagnosis have dramatically improved our ability to monitor fetal growth and detect any abnormality through the measurement of biometric markers.

FGR may be symmetrical or asymmetrical (Boger 2003). The former is a growth restriction that, usually, arises in the second trimester of pregnancy. It is defined harmonic, because it affects all organs (included brain) and may be limited to genetic anomalies of the fetus, infectious diseases of the mother, or toxic agents. Instead, asymmetrical restricted growth usually arises in third trimester of pregnancy and it can be associated to various pathological conditions of the mother, determining an alteration of placental function with subsequent reduction of fetal oxygen and nutrients. In this case, the perfusion to certain organs is preserved, with the growth of the body being lower compared to that of the skull. FGR can be associated with oligohydramnios, probably due to a reduced fetal renal perfusion, secondary to the redistribution of blood in the fetus.

2.4.2 Fetal and perinatal complications

FGR is a crucial factor for perinatal mortality and morbidity, including ‘unexplained’ stillbirth (Blair and Stanley 1990, Schwarze, Gembruch et al. 2005, Akturk, Onal et al. 2007). Fetal complications due to FGR include intrapartum distress,hypoxia, asphyxia, and fetal demise. Neonatal complicationsinclude meconium aspiration, metabolic and hematological disturbances,cognitive dysfunction, and cerebral palsy (Hack and Fanaroff 2000). Recent epidemiological studies report a considerable proportion of permanent neurological

sequels to peri-/intranatal asphyxia, leading to severe hypoxic-ischemic encephalopathy, as the single most frequent cause for cerebral palsy.

2.4.3 FGR and long-term morbidity

In the last 40 years, it has been demonstrated that FGR is associated with an increased long-term morbidity (Hack and Fanaroff 2000). In addition, several investigators are, today, reported a possible association between low weight at birth and higher risk of metabolic dysfunctions in adult age, including which obesity, diabetes mellitus, premature atherosclerosis and cardiovascular diseases (CVD) (Phipps, Barker et al. 1993) (Goodfellow, Bellamy et al. 1998) (Leeson, Kattenhorn et al. 2001) (Adabag 2001) (Lauren, Jarvelin et al. 2003) (McNeill, Tuya et al. 2004). Specifically, newborns with a reduced fetal growth present a thicker aortic wall (Skilton, Evans et al. 2005) as well as higher blood levels of cortisol, triglyceride and low density lipoprotein (LDL) (Hossain, Islam et al. 2006).

2.4.4 Established etiological factors

A low weight at birth can be due to maternal factors. They are alcohol, smoking, drugs, serious anemia, undernutrition, cardiac and renal diseases, pre-gestational diabetes, celiac disease, uterine malformations, placenta previa, chronic and gestational hypertension, and infections (citomegalovirus, toxoplasmosis). Fetal factors include specific genetic background, multiple pregnancy and congenital anomalies (Cogswell and Yip 1995).

2.4.5 Emerging role of endothelium in the pathophysiology of FGR

The placenta is the ‘lung of the fetus’. In the placenta, oxygen from maternal red cells is exchanged for carbon dioxide coming from the fetal circulation. Deoxygenated fetal blood reaches the chorionic villi via the two umbilical arteries and oxygenated fetal blood returns to the fetus via the single umbilical vein. The partial pressure of oxygen in the fetal blood is much lower than in the adult. However, due to the high fetal cardiac output, organ blood flow, hemoglobin concentration and affinity of fetal hemoglobin for oxygen, the fetus normally has a surplus of oxygen available for its energy requirements(Clerici, Luzietti et al. 2001)

Fetal hypoxemia may be the result of different feto-maternal pathophysiological processes which can produce completely different fetal hemodynamic modifications, not only in relation to the quality but particularly in relation to the chronology of the hemodynamic events.

Despite the presumptive contribution of several factors, no underlying cause can be identified in at least 40% of children.

Several mechanisms are involved in the beginning of the processes which lead to fetal hemodynamic changes, from adaptation to decompensation, during hypoxemia: feto-maternal immunologic tolerance alterations, failure of the endothelial vasodilator tone control, reduction of maternal plasmatic expansion, increased maternal blood viscosity at a low shear rate, inappropriate trophoblastic invasions with histological, morphological and functional placental alterations and others. All these processes are involved in the hemodynamic alterations in both uterine and umbilical arteries which characterize fetuses with fetal growth restriction (Jauniaux, Jurkovic et al. 1992) (Trudinger, Cook et al. 1987).

However, fetal antepartum oxygen deficiency is mostly due to placental vascular insufficiency, and it is important to point out that fetal hypoxemia-acidemia is part of the terminal pathway starting from placental functional and structural alterations through to fetal growth restriction, potentially leading to fetal damage or fetal death.

In detail, the arteriolar vasoconstriction could be followed by histological modifications with thickening and/or degeneration of the vascular wall (Fok, Pavlova et al. 1990). Additionally, a reduction of vascular density and stromal capillaries in placental villi suggests that an altered vascular structure may underlie some of the functional alterations of placental blood flow observed during FGR (Arroyo and Winn 2008). The primary cause for these alterations is yet to be conclusively established, but a deranged synthesis of endothelial-derived factors, including abnormal levels of angiogenic and anti-angiogenic growth factors or impaired regulation of oxygen exchanges, has been proposed to be responsible for many pathophysiological features of FGR (Arroyo and Winn 2008). Overall, increasing evidence indicates that an altered function of feto-placental endothelial cells may play a prominent role in the pathophysiology of FGR. However, the exact mechanisms of any such alteration remains to be determined.

Similar factors may be involved in the defective vascular remodeling on the maternal side observed in FGR pregnancies (Arroyo and Winn 2008). In fact, it is presently known that an abnormal utero–placental circulation can be associated with an insufficient transplacental amino acid transport and a reduced passage of glucose and oxygen (Thureen, Trembler et al. 1992) (Ross, Fennessey et al. 1996, Galan, Hussey et al. 1998), caming ultimately of local hypoxia.

During normal pregnancy, uterine and systemic vascular function changes (Thaler, Manor et al. 1990). In early pregnancy, the trophoblast invades the inner third part of the myometrium as early as after 8 weeks of gestation and migrates through the entire length of the spiral arteries; this phase is completed by the 20th week (Pijnenborg, Dixon et al. 1980). In the process, the spiral uterine arteries lose their elastic layer and are transformed into markedly dilated uteroplacental arteries (Papageorghiou, Yu et al. 2001) (Papageorghiou, Yu et al. 2002) (Prefumo, Guven et al. 2004). These morphological changes are essential for the normal progress of pregnancy, being necessary for the increasing need for feto-maternal exchanges with advancing gestation. In FGR and pre-eclampsia (PE) the physiological modifications of the spirals vessels seem to be limited to the intra-decidual district and involve a small percentage of vessels, determining an elevated stiffness of the uterus-placental vascular bed. In particular, asymmetrical FGR is associated with an impairment of uteroplacental blood flow that may be reduced of up to 50% as a result of impaired trophoblast invasion of spiral arteries. Hence, blood flow is restricted to the intervillous space (Trudinger, Cook et al. 1987) (Krebs, Macara et al. 1996) (Salafia and Parke 1997, Galan, Hussey et al. 1998).

2.4.6 Pathophysiology of the fetal response to chronic hypoxemia

When the structural and functional placental alterations appear and/or increase, the fetus adapts itself to this situation with decreased growth, alterations in behavior (i.e. decrease in the episodes of body movements) and hemodynamic changes in order to maintain the supply of oxygen and substrates for tissues with active metabolism such as the brain, heart and adrenals (Mari and Deter 1992). Only when the obstruction of placental vessels is greater than 60% is there a detectable and clear alteration in the umbilical artery velocity waveform profile (Trudinger, Cook et al. 1987). Thus, when a particular level of pO2 is reached, there is a redistribution of the fetal blood flow. These hemodynamic modifications, which are known as the ‘brain

sparing effect’ and which produce a ‘fetal hemodynamic centralization’, are thought to be protective against hypoxic insult and consist of vasodilatation with an increase in blood flow in the fetal structures which are most sensitive to hypoxemia (such as the brain, adrenals and coronary arteries) and a decrease in the blood supply in the peripheral vascular districts such as pulmonary, intestinal, cutaneous, renal and skeletal vessels (Weiner, Farmakides et al. 1994). These changes in arterial perfusion are mediated by neuronal stimulation, either directly through stimulation of the vagal center or through chemoreceptors in the aorta and in the carotid arteries. If the uteroplacental vascular bed alterations persist, this produces a further increase in the impedance to flow in the umbilical artery and in the fetal aorta and, mainly as a result of the hypoxemia, in the renal artery. Moreover, these factors cause a further increase in the hypoxemic fetal status, balanced by a more pronounced fetal blood flow redistribution with lowest impedance to flow values in the cerebral vessels. This ‘centralization of blood flow’ influences cardiac hemodynamics with a decreased left ventricle afterload due to the cerebral vasodilatation and an increased right ventricle afterload due to the systemic vasoconstriction. This phase is characterized by the extreme response of the fetus to hypoxemia, which may lead to the de-compensatory phase.

The last phase is characterized by the impairment of fetal cardiac function.

Due to the persistent severe hypoxemia and the consequent polycythemia and increased blood viscosity, there is an impairment of fetal cardiac contractility, which is the most important factor leading to the terminal de-compensatory phase. The impairment of cardiac function causes a decrease in the cardiac afterload and an increase in the cardiac preload, leading to an increase in the atrioventricular gradient and abnormal ventricular filling with an increase in venous pressure beyond the inferior vena cava, hepatic and venous ductus circulation throughout the umbilical vein blood flow. Moreover, during this stage, the reduced cardiac output and the high blood viscosity also cause a reduction in the cerebral perfusion, leading to the disappearance of the so-called ‘brain sparing’. The disappearance of the latter may also be induced by a mechanical mechanism brought about by the edema caused by the brain damage from the hypoxic insult (Clerici, Luzietti et al. 2001). Sonography and, particularly, Doppler ultrasound technologies can help the obstetrician in the evaluation of the antepartum well-being of the fetus, and of fetal hemodynamic adaptations to different maternal and fetal pathophysiological conditions leading to fetal hypoxemia.

2.4.7 FGR and NO SYSTEM 2.4.7.1 NO and FGR

Conflicting results are available on feto-placental NO production in FGR. FGR has been linked to a significant fall in maternal and umbilical NO (Tranquilli, Giannubilo et al. 2004) (Ying, Chen et al. 1999) and cGMP concentration (Schiessl, Strasburger et al. 2006) as well as to significant alterations in placental L-arginine transport (Casanello and Sobrevia 2002). However, other studies have reported high NO plasma levels and increased eNOS expression (Izumi, Makino et al. 1995, Norris, Higgins et al. 1999, Benedetto, Marozio et al. 2000, Wijnberger, Krediet et al. 2001, Akturk, Onal et al. 2007) in pregnancies complicated by pre-eclampsia and/or FGR, suggesting a compensatory mechanism for the preservation of blood flow to the uteroplacental unit (Lyall, Young et al. 1995) (Di Iorio, Marinoni et al. 1997, Arroyo, Anthony et al. 2006).

In conclusion, the role of NO in normal pregnancies or in pregnancies complicated by preeclampsia or FGR is still controversial.

2.4.7.2 ADMA and FGR

During pregnancy maternal hemodynamics has been reported to be modulated by a reduction in ADMA (Maeda, Yoshimura et al. 2003). An increase in ADMA concentration in the placenta could affect a number of processes, including trophoblast motility, invasion and survival (Gagioti, Scavone et al. 2000). In addition, elevated plasma concentration of ADMA in pregnancies complicated by FGR (Fickling, Williams et al. 1993, Holden, Fickling et al. 1998, Ellis, Wennerholm et al. 2001, Savvidou, Hingorani et al. 2003) has been suggested as a potential causal factor in association with endothelial dysfunction. A compensatory upregulation of placental DDAH activity has also been reported in patients with preeclampsia (Siroen, Teerlink et al. 2006). However, recent reports do not confirm a role for ADMA in the determination of FGR (Maas, Boger et al. 2004, Prefumo, Thilaganathan et al. 2008).

2.4.7.3 The role of L-arginine

L-arginine is an efficient NO precursor and in virtue of this action vasodilates vessels and exerts an anti-aggregatory effects on platelets, hence in improving feto-maternal blood flow. There is evidence that administration of NO donors in pregnancies complicated by hypertension and FGR improves both maternal and fetal hemodynamics, ensuring prolongation of gestation (Karowicz-Bilinska, Kowalska-Koprek et al. 2003, Sieroszewski, Suzin et al. 2004, Xiao and Li 2005). Disturbances induced by chronic inhibition of endothelium-derived NO synthesis (hypertension, FGR, proteinuria, renal glomerulus’s injury) are reversed by treatment with L-arginine (Helmbrecht, Farhat et al. 1996). These findings account for the use of NO donors in the treatment and prevention of FGR and preeclampsia and support the concept of endothelial dysfunction underlying FGR.

2.4.8 GENE PHENOTYPE AND ENDOTHELIAL FUNCTION IN FGR

Placental development can be divided into vasculogenesis, in which an initial vascular network is formed, and angiogenesis, in which the network is remodelled. Additional blood vessels are generated by sprouting, branching and differential growth of the initial vessels to form a more mature system with larger and smaller vessels (Regnault, Galan et al. 2002). There are indications that angiogenic growth factors related to VEGF (vascular endothelial growth factor) may be implicated in the trophoblast invasion. From bFGF (basic fibroblast growth factor) and VEGF, the study of angiogenesis has expanded to include many additional agonists, receptors and inhibitors, especially PlGF (placental growth factor) and its soluble receptor sFlt-1 (soluble fms-like tyrosine kinase 1) (Arroyo and Winn 2008). Particularly, an ‘anti-angiogenic state’ has been implicated as a mechanism of disease in preeclampsia, HELLP syndrome (hemolysis, elevated liver enzymes, low platelets), and for FGR (Torry, Mukherjea et al. 2003). This state appears to result from an imbalance in the production and circulating concentrations of angiogenic factors such as PlGF and VEGF and anti-angiogenic factors such as soluble VEGF receptor-1 (sVEGFR-1) and soluble endoglin (s-Eng). Recently, it has been proposed that serial determinations of the concentrations of sVEGFR-1, PlGF, and

s-Eng are more informative in assessing the risk for PE than are single measurements in the first or second trimester. For example, most patients with a subnormal increase in PlGF and increasing concentrations of s-Eng and sVEGFR-1 (10 out of 17 patients) are proven to develop preeclampsia. Longitudinal studies have demonstrated that these changes precede the development of overt disease or the delivery of an SGA neonate (Arroyo and Winn 2008).

2.4.8.2 Extracellular matrix changes in feto-placental vessels

Tissue architecture results from specific cell-cell and cell-matrix interactions. The exact regulation of placentation is still to be elucidated. The invasion of the cytotrophoblast is regulated by secretion of proteases, in particular matrix-metalloproteinases (MMPs). MMPs are a family of zinc-requiring enzymes, directed against extracellular matrix (ECM) components (e. g. gelatin, collagen, elastin, laminin, fibronectin). They can be divided in four groups, the collagenases [interstitial collagenase (MMP-1), neutrophil collagenase (MMP-8), [human collagenase] (MMP-13)], type IV collagenases or gelatinases [gelatinase A (MMP-2), gelatinase B (MMP-9)], stromelysins [stromelysin-1 (MMP-3)] and the membrane-type matrix-metalloproteinases (MT-MMP) (Kinzler, Smulian et al. 2005). It has been demonstred that the tissue inhibitors (TIMPs) inhibit the activity of the MMPs by binding to the highly conserved zinc-binding site of active enzymes. MMPs play a key role in tissue remodeling in both normal and pathological processes. Both MMP2 (gelatinase A/72 kDa type IV collagenase) and MMP9 (gelatinase B/92 kDa type IV collagenase) genes are expressed in first trimester trophoblast cells with MMP2 abundance decreasing and MMP9 abundance increasing over time (Merchant, Crocker et al. 2004). It is not known whether MMP activity in placental bed is compromised in pregnancies which develop FGR in later trimesters (Huisman, Timmer et al. 2004). It has also been demonstrated that the fibrillar collagens are the major extracellular component responsible for tissue structure. They aggregate to form banded fibrils of various diameters. Stromal cell differentiation is associated with loss of collagen VI, which facilitates invasion through the interstitial spaces of the deciduas. Various conditions can effect collagen and proteoglycan production within the extracellular matrix. Hypoxia is one such condition promoting matrix remodelling (Bischof,

Meisser et al. 2000). As a result, the ECM of fetal blood vessels may undergo architectural changes and this can predispose to altered vessel function (Kinzler, Smulian et al. 2005).

2.4.8.3 Adhesion molecules expression in the placental bed

Cell adhesion molecules play a key role in the biological process of angiogenesis and morphogenesis and in the pathophysiology of a variety of diseases, including thrombosis, inflammation, ischaemia and reperfusion injury, transplant rejection and carcinogenesis. Adhesion molecules are divided into four groups according to their structure: integrins, immunoglobulin-like proteins [immunoglobulin gene superfamily, such as intercellular cell adhesion molecule-1, intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1, CD106 (VCAM-1) and platelet-endothelial cell adhesion molecule-1, CD31 (PECAM-1)], selectins and cadherins.

The trigger which initiates endothelial cell damage and dysfunction and the nature of the underlying vascular pathology are not well defined and the studies of these adhesion molecules in women with FGR have produced conflicting results. Exposure of endothelial cells to fetal plasma from pregnancies with umbilical placental vascular disease seems to increase the expression of mRNA for the cell adhesion molecules ICAM-1 and PECAM-1 compared with normal pregnancy. These findings support the view that placental vascular disease is induced by factor(s) released into the fetal microcirculation, and is associated with endothelial cell activation (Wang, Athayde et al. 2002).

2.5. PRE-NATAL METABOLIC RISK OF ADULT CARDIOVASCULAR DISEASES

Development in utero, from conception to birth, is largely predetermined by the genetic profile. A relatively new concept is that of metabolic ‘programming’ or ‘imprinting’, which suggests that factors encountered in utero may also determine the developmental pattern and individual susceptibility to diseases later in life (Langley-Evans, Sherman et al. 1999). Programming is defined as a permanent response to an insult or stimulus, experienced during a critical, or vulnerable, period of development (Leeson, Kattenhorn et al. 2001). The developing fetus goes through a number of critical developmental periods when organogenesis

and differentiation take place. Any programming of an organism or tissue may be regarded as the consequences of an adaptation that is necessary to survive an insult. Adaptations to ensure survival may, at critical points in development, result in long-term or permanent changes to organ morphology, metabolic functions, endocrine functions and physiology.

Programmed alterations in metabolic or physiologic functions may be of long-term benefit to the organism in addition to accomplishing short-term survival. Some programmed changes may, conversely, compromise the well-being of the individual in life later, increasing susceptibility to disease (Langley-Evans, Sherman et al. 1999).

2.5.2 Fetal origins hypothesis

Barker´s group originally claimed that a substantial part of the risk of cardiovascular pathologies, type 2 diabetes and hypertension is established during development. Has been suggest that this reflects two widespread biological fenomena, i.e. developmental plasticity and compensatory growth (Barker, Eriksson, et al. 2002) .

The developmental plasticity, in a changing environment, enables the production of phenotypes that are better matched to their environment than would be possible by the production of the same phenotype in all environments.

On the other hand, there are a number of other possible processes by which, in humans, undernutrition and small size at birth followed by rapid childhood growth could lead to CVD and type 2 diabetes in later life. One suggestion is that a higher rate of cell division causes more rapid shortening of the protective ends of the chromosomes (telomeres) and hastens cell death and organ degradation. Additionally, rapid weight gain may lead to an unfavourable body composition. Babies that are small and thin at birth lack muscle, a deficiency which will persist as the critical period for muscle growth occurs in utero and there is little cell replication after birth. If they develop a high body mass during later childhood they may have a disproportionately high fat mass in relation to lean body mass, which will lead to insulin resistance (Barker 1998).

Summarizing, a specific answer to why undernutrition lead to disease in later life is that people who were small at birth are vulnerable to later disease through three kinds of process (Barker 1998).

First, they have fewer cells in key organs, such as the kidney. One theory holds that hypertension is initiated by the reduced number of glomeruli found in people who were small at birth. A reduced number necessarily leads to increased blood flow through each glomerulus. Over time this hyperfiltration is thought to lead to the development of glomerulo-sclerosis which, combined with the loss of glomeruli that accompanies normal ageing, leads to accelerated age-related loss of glomeruli, and a self-perpetuating cycle of rising blood pressure and glomerular loss.

Another process by which slow fetal growth may be linked to later disease is in the setting of hormones and metabolism. An undernourished baby may establish a “thrifty” way of handling food. Insulin resistance, which is associated with low birthweight, may be viewed as persistence of a fetal response by which blood glucose concentrations were maintained for the benefit of the brain, but at the expense of glucose transport into the muscles and muscle growth.

A third link between low birthweight and later disease is that people who were small at birth are more vulnerable to adverse environmental influences in later life.

2.5.3 ADMA and CVD later in life

High ADMA is an marker of endothelial dysfunction (Fard, Tuck et al. 2000, Achan, Broadhead et al. 2003). In particular, through measurement of plasma ADMA levels in 116 clinically healthy humans without overt signs of coronary or peripheral arterial disease, Miyazaki and co-workers have established a significant relationship of ADMA concentration with age, mean arterial blood pressure, and glucose tolerance. In a multivariate regression analysis, a significant relationship between ADMA and intima-media thickness of the carotid artery has also been found found (Miyazaki, Matsuoka et al. 1999). Furthemore, a group of investigators from the Netherlands (Nijveldt, Teerlink et al. 2003) have sought to identify novel risk factors for survival during ICU treatment. Among all biochemical markers of organ function and disease risk measured in this study, ADMA was the single factor with highest predictive power. In fact, patients with elevated ADMA levels showed a 17-fold higher risk of complications. ADMA may, thus, play a role in the