European Journal of Obstetrics & Gynecology and Reproductive Biology 113 (2004) 17–20
Amniotic vascular endothelial growth factor (VEGF) and nitric oxide (NO) in women with subsequent preeclampsia Andrea L. Tranquillia,*, Valeria Bezzeccheria, Stefano R. Giannubiloa, Caterina Scagnolia, Laura Mazzantib, Giuseppe G. Garzettia a
Department of Obstetrics and Gynecology, University of Ancona, Salesi Hospital, via Corridoni 11, 60123 Ancona, Italy b Department of Biochemistry, University of Ancona, Salesi Hospital, via Corridoni 11, 60123 Ancona, Italy Received 11 November 2002; received in revised form 29 April 2003; accepted 23 May 2003
Abstract Objective: To assess whether amniotic fluid concentrations of nitric oxide (NO) and vascular endothelial growth factor (VEGF) in early pregnancy correlate to subsequent preeclampsia. Study design: We performed a retrospective study to assess VEGF and NO on the second trimester amniotic fluid of 15 healthy women, and 15 women who subsequently developed preeclampsia. Results: In women with subsequent preeclampsia, both VEGF (213:19 78:42 pg/ml) and NO concentrations (4:31 1:02 mmol/mg creatinine) were significantly lower than healthy controls (VEGF 255:05 88:66 pg/ml; NO 5:02 1:57 mg/mg creatinine; P < 0:05) Conclusions: Our findings suggest that reduced VEGF may be responsible, at least in part, for the impaired vascular development which occurs in preeclampsia. Low concentrations of VEGF and NO in the second trimester may represent an impaired stimulus to vascular formation and endothelial regulation that induce placental disease and preeclampsia. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: NO; VEGF; Preeclampsia
1. Introduction Preeclampsia, which affects 10% of all first pregnancies, is a multisystem disorder characterized by hypertension and proteinuria. It is among the leading causes of fetal and maternal morbidity and mortality. Although the etiology is unclear, there is accumulated evidence for a pathogenic model of preeclampsia whereby a deficiency in trophoblastic invasion of the placental bed leads to a poorly perfused fetoplacental unit. During normal pregnancy, uterine and systemic vascular function changes drammatically [1]. In early pregnancy, the trophoblast invades the inner third part of the myometrium as early as 8 weeks of gestation and migrates through the entire length of the spiral arteries; such process completes in the 20th week [2]. In this process, the spiral uterine arteries lose their elastic layer and are transformed into markedly dilated uteroplacental arteries. These morphological changes are essential, as they provide for the everincreasing demand imposed on the maternal placental cir* Corresponding author. Tel.: þ39-071-5962214; fax: þ39-071-36575. E-mail address:
[email protected] (A.L. Tranquilli).
culation by the advancing gestation. The normal angiogenesis (i.e. the formation of vessels from already preexisting ones) is regulated by oxygen partial pressure and by the growth factors, such as the vascular endothelial growth factor (VEGF) [3]. The relaxant effect on arteriolar tone is achieved through a complex interaction of many vasoactive substances. It is described that increased endothelial production of vasodilators, such as endothelium derived nitric oxide (NO), during pregnancy may be primarily responsible for the observed loss of vascular responsiveness to otherwise potent vasoconstrictors. Any imbalance in the equilibrium of the vasoactive substances may compromise the normal angiogenesis and may lead to pathological conditions. The purpose of this study was to determine whether amniotic fluid concentrations of NO and VEGF in early pregnancy correlate to subsequent preeclampsia.
2. Materials and methods By the data of amniotic fluid bank of our Institution, we retrospectively recruited 15 women who subsequently
0301-2115/$ – see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0301-2115(03)00369-5
A.L. Tranquilli et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 113 (2004) 17–20 300 Normotensive Preeclamptic
250 200 pg/ml
developed mild preeclampsia (median age: 38.5 years, range 34–40; median parity: 1, range: 1–3), matched for age and gestation with 15 women who had phisyologic pregnancy and who served as controls (median age: 37.8 years, range 33–41; median parity: 1, range: 1–3). All women had undergone amniocentesis for maternal age and were normotensive and non-proteinuric at moment of sampling (16 weeks’ gestation as assessed by a first trimester ultrasound). Inclusion criteria were—absence of the following conditions: history of chronic hypertension, renal and cardiac disease, diabetes mellitus, thyroid and immunologic diseases and congenital or acquired thrombophilic disorders. Fetal karyotype resulted normal in all cases. From amniotic fluid, samples of nitrites/nitrates (NOx), which are the metabolites of nitric oxide, and Vascular Endothelial Growth Factor concentrations were assessed. Mild preeclampsia was defined, strictly according to internationally approved criteria [4], as an elevated blood pressure (>140/90 mmHg) for at least two consecutive readings 6 or more hours apart, in association with significant proteinuria (>300 mg/24 h), occurring at a gestational age greater than 20 weeks. The protocol of the study was approved by the University’s Ethical Committee. Informed consent was obtained from all of the patients.
150 100 50 0
amniotic VEGF Fig. 1. VEGF concentrations (pg/ml) on midtrimester amniotic fluid from normotensive women and women who subsequently developed preeclampsia. (Student’s t-test P < 0:05).
6 Normotensive Preeclamptic
5 mmol/mg creat.
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4 3 2
2.1. Biochemical assay 1
Samples of amniotic fluid by amniocentesis were centrifuged immediately after collection at 1800 g for 15 min, at the temperature of 4 8C; the supernatant was divided into aliquots and stored at 80 8C until laboratory assay. All samples were transported in ice to the laboratory of our Institute of Biochemistry. Since the final products of NO in vivo are nitrite (NO2) and nitrate (NO3) (which are commonly referred as NOx) and the relative proportion of NO2 and NO3 is variable and cannot be predicted with certainty, the best index of total NO production is the sum of both NO2 and NO3. We have assayed NOx in amniotic fluid samples (80 ml) using a commercial kit (Cayman Chemical, Ann Arbor MI, USA). To ensure that differences among groups were not due to a different rate of fetal urine production, the amniotic fluid values of nitrate were normalized for creatinine. The assay of VEGF was performed by quantitative sandwich enzyme immunoassay technique (ELISA), using the kit Quantikine (R&D Systems Human VEGF Immunoassay), Results were expressed as mean and standard deviation. Student’s t-test was used to compare results. Statistical significance was assessed at P < 0:05.
3. Results In normal pregnancies, amniotic fluid concentrations of VEGF were 255:05 88:66 pg/ml and of NOx were 5:02 1:57 mg/mg creatinine. In women with subsequent
0
amniotic NOx Fig. 2. NO concentrations (mmol/mg creatinine) on midtrimetster amniotic fluid from normotensive women and women who subsequently developed preeclampsia. (Student’s t-test P < 0:05).
preeclampsia, both VEGF (213:19 78:42 pg/ml P < 0:05) and NOx amniotic fluid concentrations (4:31 1:02 mg/mg creatinine P < 0:05) were significantly lower than healthy controls (Figs. 1 and 2).
4. Comment Nitric oxide involvement in the pathogenesis of preeclampsia is still controversial. Morris states that a relative deficiency of placental nitric oxide in pregnancies complicated by fetal growth retardation and preeclampsia may contribute to the development of the high fetoplacental circulation impedance found in these conditions [5]. Cockell and Poston [6] hypothesized that enhanced responses to shear stress, which leads to enhanced nitric oxide release, may play an important role in the adaptation of the maternal circulation to pregnancy. Studies using an inhibitor of nitric oxide synthase [7] or a nitric oxide donor [8] also support that compromised nitric oxide production is likely to be a
A.L. Tranquilli et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 113 (2004) 17–20
common feature of preeclamptic women with intrauterine growth restriction and consequent vasoconstriction causes a decline in uteroplacental circulation as compared with the elevation of blood pressure. Others Authors state that concentrations of NO metabolites are higher in the amniotic fluid of preeclamptic patients [9,10]. Our data show a decrease in NO metabolites and VEGF in a second trimester amniotic fluid in women who subsequently developed preeclampsia but were normotensive at moment of sampling, therefore in early phase of trophoblastic development. It is well known that insufficient trophoblast invasion of maternal spiral arteries contributes to the development of preeclampsia. Histological examination of placental bed biopsies from preeclamptic women demonstrates trophoblast proliferation, but limited migration into superficial decidua [11]. Genbacev et al. [12] have shown that cytotrophoblast tissues are extremely sensitive to oxygen pressure and that oxygen may be the signal trigging these cells to reduce cell division and to differentiate. The rise of PO2 may be the trigger for the trophoblast to change to an invasive extravillous trophoblast, thus the secondary wave of trophoblast invasion of the maternal spiral arterioles, establishing a high flow, determines low impedance uteroplacental circulation. Pregnancies complicated by the multiorgan failure of preeclampsia are associated with poor placental growth and inadequate physiologic changes in the vasculature of the placental bed. Blood vessel differentiation and growth results from the interaction of different proteins, including cell adhesion molecules, extracellular matrix components, angiogenic growth factor and their receptors. VEGF is the most common mediator of angiogenesis; it has important effects on endothelial cells increasing cell proliferation, permeability and nitric oxide production. Failure to achieve these adaptations may result in reduced feto-placental perfusion, which develops during disease states such as preeclampsia [13], intrauterine fetal growth restriction [14] or fetal death. On the other hand, nitric oxide is responsible of the maintenance of low resistance in placenta, allowing an adequate oxygen/carbon dioxide and nutrients exchange between mother and fetus. An involvement of nitric oxide in the regulation of angiogenesis has recently been suggested; moreover, a variety of studies indicate that NO plays a direct role in the mechanism of action of VEGF. Such interactions are not completely defined, although in some situations, such as ischemia, they share sinergic effects. Furthermore, NO synthase inhibitors block the vasodilating effect of VEGF in vitro. In fact others investigations support a role for NO and VEGF interactions, with a hierarchic dependence of VEGF from NO: if NO concentrations are reduced VEGF may not adequately accomplish its functions [15]. Those researchers outline a possible stimulating action of NO on the ability of VEGF to promote the angiogenesis, mediated by GMPc concentrations: the NO synthase/GMP cyclase cascade seems to be the common pathway through
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which NO and VEGF trigger mytogenesis in endothelial cells stimulated by chemical and physical agents. All these considerations make clear that an imbalance in the production of NO, VEGF or both, in early pregnancy may lead to altered placental development, altered placental function, and related diseases. From this background, it may be hypothesized that VEGF, besides its action on vascular permeablilty and proliferation, has a vasodilating effect mediated by the NO synthase through an increase in endothelial intracellular calcium. Our results suggest that, in a very early phase of gestation, low concentrations of NO may represent a reduced stimulus to angiogenesis that may lead to a subsequent altered placentation. The abnormal action may be direct, since NO concentrations are not sufficient to trigger angiogenesis, or indirect, since VEGF itself is reduced, or its action is less effective because of the low NO concentrations. Pathologic conditions can be the result of an early onset of placenta hyperoxia with decreased expression of VEGF and NO, from which come the observed decreased branching angiogenesis, the failure of non-branching angiogenesis within the terminal villi and the disturbances in the rate of trophoblast growth. A possible limitation of our study is that, since we recruited the women who performed amniocentesis for maternal age, the population was older than 35 years and this is per se a risk factor of preeclampsia. Even if the cases were matched for age and parity with controls our conclusions may be supposed, to date, only for this age period. In conclusion, in normal early pregnancy NO and VEGF are important stimuli for the complex process of angiogenesis, which is essential to an adequate development of the placenta. When NO is early reduced, the outcome of pregnancy may be unfavorable. The low concentrations of NO and VEGF we observed in amniotic fluid from the early second trimester may be the sign of an imbalance of placental vascularization and altered endothelial function, both responsible for the subsequent occurrence of preeclampsia. References [1] Thaler I, Manor D, Itskovitz J, Rottem S, Levit N, Timor-Tritsch IE, et al. Changes in uterine blood flow during human pregnancy. Am J Obstet Gynecol 1990;162:121–5. [2] Pijnenborg R, Dixon G, Robertson WB, Brosens I. Trophoblastic invasion of human decidua from 8–18 weeks of pregnancy. Placenta 1980;1:3–19. [3] Cy C. Vascular endothelial growth factor: possible role in fetal development and placental function. J Soc Gynecol Invest 1997;4: 169–77. [4] Brown MA, Lindheimer MD, de Swiet M, Van Assche A, Moutquin JM. The classification and diagnosis of the hypertensive disorders of pregnancy: statement from the International Society for the Study of Hypertension in Pregnancy (ISSHP). Hyper Pregnancy 2001; 20:9–14. [5] Morris NH, Sooranna SR, Learmont JG, Poston L, Ramsay B, Pearson JD, et al. Nitric oxide synthase activities in placental tissue from normotensive. Br J Obstet Gynaecol 1995;102:711–4.
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