Nitric oxide synthase activities in placental tissue from normotensive, pre-eclamptic and growth retarded pregnancies

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British Journal of Obstetrics and Gynaecology September 1995, Vol. 102, pp. 71 1-714

MATERNAL-FETAL MEDICINE

Nitric oxide synthase activities in placental tissue from normotensive, pre-eclamptic and growth retarded pregnancies N I C H O L A H. S MORRIS,WellBeing Training Fellow, * S U R E NR. S O O R A N NSenior A Biochemist, J O N A T H A NG. L EA R M O N T Research Registrar, ** L U C I L L A POSTON Professor (Fetal Research). * B R U C E R A M S E Y Registrurl Lecturer, * * * J E R E M Y D. P E A R S O Professor N (Vascular Biology) * P H I L I PJ. STEERProfessor (Obstetrics and Gynaecology) ‘Reproductive Vascular Group, Academic Department of Obstetrics and Gynaecology, Chelsea and Westminster Hospital, London; *’ U M D S Academic Depurtment of Ohstetrics and Gynarcologv. St Thomas’ Campus, London: *** Vascular Biology Reseurch Centre, King’s College London

ABSTRACT Objective To measure nitric oxide synthase activity in tissues from the placenta, placental bed and umbilical cord at delivery in normal and complicated pregnancies. Design A prospective blinded study. Setting The obstetric departments of three London teaching hospitals. Subjects Samples of whole placenta, dissected stem villous arteries, umbilical cord vessels and the placental bed of the uterus were collected at delivery and assayed for nitric oxide synthase activity. Samples of placenta were taken from ten normotensive, six pre-eclamptic and eight growth retarded pregnancies, and stem villous arteries from a further seven normotensive pregnancies. Results There was minimal placental bed nitric oxide synthase activity in each group. Placental villous homogenates from pregnancies complicated by pre-eclampsia and fetal growth retardation had significantly lower activities of nitric oxide synthase than those from normotensive women with appropriately grown babies. There were no significant differences in calcium dependent or calcium independent nitric oxide synthase activities in the umbilical vein and artery in the normal or in the pre-eclamptic groups. However, there was significantly more calcium dependent than calcium independent nitric oxide synthase in the umbilical veins in all groups. Conclusions Local nitric oxide production in the placental bed of the uterus is unlikely to contribute substantially to the low resting vascular tone in the uteroplacental circulation. However, a relative deficiency of placental nitric oxide in pregnancies complicated by fetal growth retardation and pre-eclampsia may contribute to the development of the high impedance fetoplacental circulation found in these conditions. The blood vessels of the fetoplacental circulation do not have an autonomic nervous system (Reilly & Russell 1977),which means that its characteristic low resistance to blood flow is conferred either by vasoactive substances or by the anatomical development of the distal branches of the villous tree (Kingdom et a / . 1994). However, in pregnancies complicated by pre-eclampsia and growth retardation, there is not only an increase in

Correspondence: Mr N. H. Morris, University Department of Obstetrics and Gynaecology, Rosie Maternity Hospital, Robinson Way, Cambrdge CB2 25W, UK.

resistance in both the uteroplacental and fetoplacental circulations (Trudinger et a/. 1985; Campbell et al. 1986), but endothelial regulation of fetoplacental vascular tone also becomes abnormal (McLaren et a / . 1987). Conrad et al. (1993a) demonstrated that there is an increased production of the potent vasodilator nitric oxide (NO) in pregnancy, and that the placenta is the major source of synthesis. NO has now been shown in vitro and in vivo to modulate fetoplacental and uteroplacental blood flow (Myatt et al. 1992; Hull et al. 1994; Ramsey et al. 1994). Blood flow and shear stress are the predominant stimulus for NO synthesis in the fetoplacental circulation (Gude et al. 1990; Learmont et al. 1994). 71 1

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NO is synthesised from L-arginine by NO synthase which can exist in either calcium dependent (caNOS) or calcium independent (ciNOS) isoforms. caNOS has been suggested to be constitutive and ciNOS the inducible isoform of NOS (Moncada et al. 1991). In this study we have investigated the hypothesis that pregnancies complicated by pre-eclampsia and intrauterine growth retardation may be associated with an abnormality of placental or uterine placental bed NO synthesis.

Subjects and methods Placental tissue was obtained at the Chelsea and Westminster Hospital from 10 normotensive nulliparous women (mean gestational age at delivery 37.6, SD 2.0 weeks) and six nulliparous women with pre-eclampsia (34.5, SD 2.0 weeks). Placental tissue was also obtained at King’s College Hospital from eight pregnancies (five nulliparous and three multiparous women) complicated by intrauterine growth retardation (33.9, SD 1.5 weeks). Elective caesarean sections were performed on six of the normotensive group (for indications such as breech presentation), five of the pre-eclamptic and seven of the growth retarded group. The diagnosis of pre-eclampsia was based on a blood pressure of at least 140/90 mmHg recorded on two separate occasions 4 h apart, and proteinuria of at least 500 mg of total protein in 24 h. Growth retardation was defined as an abdominal circumference less than the 2.5th centile, together with Doppler studies showing raised umbilical artery pulsatility index and fetal blood flow redistribution (Vyas et al. 1990). Biopsies of the placental bed were obtained at caesarean section, as described by Brosens et al. (1977). In a separate study at St Thomas’ Hospital, stem villous vessels were dissected under a light microscope to remove the surrounding trophoblast, by one of the authors (J. G. L.) from five placentae obtained at elective caesarean section in normotensive pregnancies. All tissue samples were wrapped in aluminium foil, weighed and placed in liquid nitrogen within a few minutes of delivery and stored at -70 “C for up to six months. All the assays were carried out in the reproductive vascular laboratory at the Chelsea and Westminster Hospital. caNOS and ciNOS activities were measured as follows : tissues were homogenised in five volumes of 50 mmol/l tris HCL buffer, pH 7.0, containing 250 mmol/l sucrose, 1 mmol/l dithiothreitol, 1 mmol/l EDTA acid, 100 pg/ml phenylmethylsulphonylfluoride, 10 pg/ml soybean trypsin inhibitor and 2 pg/ml aprotinin. NOS activity was measured in the homogenates by the conversion of L[Wlarginine to L-[”Clcitrulline (Salter et al. 1991). The activity of caNOS was determined from the difference between the [“C]citrulline produced from control samples and samples containing L-NMMA. The activity of ciNOS was determined from the difference between the [“C]citrulline produced in the presence of 1.5 mmol EGTA and samples containing 1.5 mmol EGTA and L-NMMA. NOS activities were presented in nmoles of L-citrulline formed per minute per gram of protein. The Wilcoxon matched pairs signed ranks test was used to compare NOS activities in different tissues in the same

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VEIN ART ERY Fig. 2. NOS activity in the umbilical cord. Samples of umbilical vein and artery from normotensive (n = 8) and pre-eclamptic (n = 4) pregnancies were assayed for caNOS ( 0 )and ciNOS (0) activities. Significant statistical difference was seen by the MannWhitney U test between the caNOS and ciNOS activities in the normal ( P < 0.003) and the pre-eclamptic (P< 0.03) umbilical veins.

women in the normotensive and pre-eclamptic groups. The Mann-Whitney U test was applied to test the differences between the three study groups. Differences were considered significant with P < 0.05.

Results NOS activities are presented in nmoles of L-citrulline formed/min/g of protein from tissue samples of each of the three groups studied. Figure 1 shows that in normotensive pregnancies there were similar NOS activities in the placental villi, the basal plate and the stem villous vessels. There was no significant difference in placental villi NOS activity by mode of delivery. However, the total NOS activities were significantly lower in the placental bed

NITRIC OXIDE SYNTHASE ACTIVITIES I N PLACENTAL TISSUE

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Fig. 3. NOS activity in placental villous tissues. Samples of placental villous tissues from normotensive (n = 8), pre-eclamptic ( n = 6) and intrauterine growth retarded (a = 8) pregnancies were assayed for caNOS ( 0 )and ciNOS (0) activities. Significant statistical difference was seen between the normal and the preeclamptic groups for caNOS and ciNOS activities (P < 0.05 and P < OOOS), respectively, and between the normal and the intrauterine growth retarded group (P< 0.001) for the caNOS and ciNOS activities.

biopsies than in the other tissues. Figure 2 demonstrates that the predominant activity in the umbilical vein was caNOS. There was no significant difference in the distribution of each isoform in the artery, or between the pre-eclamptic and normotensive umbilical vessels. The median and ranges for caNOS and ciNOS activities in the uterine placental bed biopsies obtained from three pre-eclamptic pregnancies were 0.10 (00-0.69) and 0.54 (0.16- 1.16), respectively, and from six normotensive pregnancies were 0.16 (0.0-3.04) and 051 (0.04- 1.18), respectively. These are not significantly different, perhaps because of the small numbers of placental bed biopsies that we could obtain. In the pre-eclamptic group, in four samples of basal plate, median and ranges for caNOS and ciNOS activities were 2.82 (0.69-5.16) and 2.22 (0-36-3.34) and in six samples of placental villi 1.61 (090-6.29) and 1.64 (0.46-3.42), respectively. Figure 3 shows that the placental villi in both the preeclamptic and the growth retarded group had significantly lower NOS activities than those from the normotensive group. Discussion This study confirms that NO synthase is widely distributed in placental tissue (Morris et al. 1993; Myatt et al. 1993; Buttery el al. 1994). caNOS and ciNOS activities were much lower in the placental bed biopsies compared with the other tissues examined, suggesting that uterine placental bed NOS activity is unlikely to have physiological significance. The vasomotor theory of increased fetoplacental vascular impedance is complex. It is dependent on both vasomotor function and on anatomical changes in the villous tree (Macara et al. 1993). We found no difference in

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NOS activities in the umbilical artery or vein in vessels from either normotensive or pre-eclamptic placentae. McCarthy et al. (1993) found no reduction in basal NO synthesis in isolated small fetoplacental vessels taken from placentae with abnormal umbilical artery Doppler profiles. In contrast, Pinto et al. (1991) have shown that umbilical vessel perfusates from patients with pre-eclampsia produced less NO than perfusates from normal vessels. The measurement of plasma nitrite and nitrate as an indicator of nitric oxide production is controversial, and there are many confounding factors including diet, reduced intravascular volume and renal clearance. This may explain why Lopez-Jaramillo et al. (1992) have shown that there are reduced nitric oxide breakdown products and second messengers in the maternal compartment in pregnancy induced hypertension, whereas Davidge et al. (1994) have shown that there is increased plasma nitrate/nitrite concentration in women with pre-eclampsia. The findings of lower activities of NOS in the villous tissue from pregnancies complicated by pre-eclampsia and growth retardation may indicate that a reduction in NOS activity has a role in generating the high resistance fetoplacental circulation. NOS activities were measured in placental villous homogenates, and so it was not possible to localise the reduction in NOS activity to either placental vessels or trophoblast. In normotensive pregnancy, caNOS has been demonstrated in placental vessels from which trophoblastic tissue has been removed (Myatt et al. 1993), and in human placental syncytiotrophoblast (Conrad et al. 1993b). Immunohistochemical techniques showed caNOS to be localised to the endothelium of umbilical arteries and veins and to villous trophoblast but not to fetal vessels in the distal regions of the villous vasculature (Buttery et al. 1994). Hypoxia reduces NO production (Kim et al. 1993). Fetoplacental hypoxia, commonly associated with intrauterine growth retardation, may well result in reduced placental endothelial and trophoblast NO synthesis. The infusion of NO synthase inhibitors in pregnant rats (Molnar & Hertlendy 1992; Molnar et al. 1994) not only increased sensitivity to vasopressor agents, but caused an increase in mean arterial pressure, maternal thrombocytopenia, intrauterine growth retardation and fetal death. NO derived from the syncytiotrophoblast may well play an important role in preventing platelet activation, adhesion and aggregation within the intervillous space. A reduction in trophoblast NO synthesis may contribute to the extensive intervillous thrombus formation in preeclampsia (Benirschke & Kaufman 1990). In conclusion we feel that reduced placental NO generation may be a reflection of placental dysfunction in pregnancies complicated by pre-eclampsia and growth retardation and may contribute to the development of proteinuric hypertension and the increase in fetoplacental vascular impedance. Acknowledgement Mr N. H. Morris was supported by a grant from WellBeing.

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Received 8 August 1994 Accepted I2 April 1995

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