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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 6 2318-2323
Copyright © 2000 by The Endocrine Society

Original Studies

Expression and Relationship between Endothelin-1 Messenger Ribonucleic Acid (mRNA) and Inducible/Endothelial Nitric Oxide Synthase mRNA Isoforms from Normal and Preeclamptic Placentas

Maddalena Napolitano, Fiorella Miceli, Angelica Calce, Alessandra Vacca, Alberto Gulino, Rosanna Apa and Antonio Lanzone

Department of Experimental Medicine and Pathology, Università La Sapienza (N.M., C.A., V.A., G.A.); Department of Obstetrics and Gynecology, Università Cattolica del Sacro Cuore (M.F., A.R.), 00168 Rome; OASI Institute for Research (L.A.), 94018 Troina; and Neuromed Institute (G.A.), 86071 Pozzilli, Italy

Address all correspondence and requests for reprints to: Antonio Lanzone, M.D., Department of Obstetrics and Gynecology, Università Cattolica del Sacro Cuore, Largo A. Gemelli 8, 00168 Rome, Italy. E-mail: SARA.LANZONE{at}TIN.it


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Preeclampsia is a mainly vascular disease of pregnancy, probably caused by an imbalance between vasodilator and vasoconstrictor agents that results in generalized vasospasm and poor perfusion in many organs. Among these factors, endothelin-1 (ET-1), a potent vasoconstrictor, is highly increased in preeclamptic women, while nitric oxide (NO), a vasodilator of human utero-placental arteries, is reduced in the same patients. The present study was designed to investigate the interactions between ET-1 and the NO system in the feto-placental unit; to this purpose we also examined the messenger ribonucleic acid (mRNA) expression of ET-1, inducible NO synthase (iNOS), and endothelial NOS (eNOS) in human cultured placental trophoblastic cells obtained from preeclamptic (PE) and normotensive (NT) pregnancies. We also studied whether exogenous ET-1 may affect the expression of iNOS and eNOS in human placental trophoblastic cells. Interestingly, by Northern blot analysis we observed an increased ET-1 mRNA expression level in PE trophoblastic cells compared to NT trophoblastic cells. Furthermore, exogenous ET-1 (10-7 mol/L) was able to up-regulate its own mRNA expression in both NT and PE trophoblastic cells. iNOS and eNOS mRNA expression was then detected, by semiquantitative PCR, in both NT and PE trophoblastic cells. PE trophoblastic cells expressed lower iNOS mRNA levels compared with NT pregnancies. On the contrary, eNOS mRNA expression was higher in PE trophoblastic cells than in NT cells. Moreover, in the presence of ET-1 we observed a decrease in iNOS and an increase in eNOS mRNA expression levels in both NT and PE trophoblastic cells compared with the respective untreated cells.

In conclusion, we demonstrate that ET-1 expression is increased in PE cells, whereas iNOS, which represents the main source of NO synthesis, is decreased; conversely, eNOS expression is increased. Finally, ET-1 is able to influence its own as well as NOS isoform expression in normal and PE trophoblastic cultured cells. These findings suggest the existence of a functional relationships between ET(s) and NOS isoforms that could constitute the biological mechanism leading to the reduced placental blood flow and increased resistance to flow in the feto-maternal circulation, which are characteristic of the pathophysiology of preeclampsia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PREECLAMPSIA (PE), a hypertensive disorder unique in pregnancy, complicates approximately 5–7% of pregnancies and is a leading cause of fetal growth retardation, indicated premature delivery, and maternal death (1, 2). It is characterized by proteinuria, edema, platelet aggregation, and increased vasoconstriction, leading to maternal hypertension and reduced utero-placental blood flow. Increasing evidence indicates endothelial cell injury or dysfunction as the primary pathophysiological mechanism leading to PE (3); the endothelial damage may be due to an imbalance between vasodilator and vasoconstrictor agents, which, in turn, may result in the generalized vasospasm and poor perfusion seen in many organs (4, 5). It has been reported, using Doppler flow velocimetry waveforms, that resistance to blood flow increases in the feto-placental vascular tree in pregnancies complicated by preeclampsia and/or intrauterine growth retardation (IUGR). Furthermore, pregnancy-induced hypertension is characterized by widespread vasoconstriction (6, 7). It has been proposed that utero-placental vascular resistance is controlled by factors released by the feto-placental unit into maternal blood (8, 9). Several substances have been suggested to be involved in such pathological events.

Endothelin-1 (ET-1) is a potent vasoconstrictor, whereas nitric oxide (NO) is a vasodilator of human utero-placental arteries; they may mutually modulate their production and/or activity by regulating each other (10, 11). Human ET-1 is synthesized as a 212-amino acid prepro-ET-1 that is cleaved by a specific endopeptidase into a 38-amino acid big ET-1. Big ET-1 is subsequently cleaved into mature ET-1 by an ET-converting enzyme. ET-1 has been shown to be part of a family of three peptides, ET-1, ET-2, and ET-3 (12). Among them, ET-1 is known to be produced by endothelial cells (13); however, lately amniotic and endometrial cells have also been shown to be able to synthesize it, and ET-1 messenger ribonucleic acid (mRNA) has been found in human placental fibroblasts and trophoblasts (14, 15, 16, 17). In addition to its direct vasoconstrictive effect on vascular smooth muscle cells, low concentrations of ET-1 have endothelium-mediated vasodilator effects that induce NO release, suggesting that it acts in a paracrine manner (18). Interestingly, the ET-1 concentration is increased in women with preeclampsia (19, 20, 21).

NO results from the enzymatic action of NO synthase (NOS), which converts L-arginine in the presence of oxygen to L-citrulline and NO (22). Three isoforms of NO synthase are known: nNOS, iNOS, eNOS, two of which are constitutively expressed in neuronal cells (nNOS) and endothelial cells (eNOS or type III) and require calcium/calmodulin for their activity, whereas the type II (iNOS) isoform is induced by a variety of cytokines and is calcium/calmodulin independent (23). Other differences between the NOS isoforms are that constitutive isoform (eNOS) produces picomoles of NO and is characterized by a short-lasting release, whereas the inducible isoform (iNOS) produces nanomoles of NO and is characterized by a long-lasting release (24). In the human feto-placental circulation, NO appears to contribute to the maintenance of low vascular resistance and to attenuate the action of vasoconstrictors such as ET-1 (25, 26, 27). Biochemical characterization showed the presence of eNOS in the feto-placental vasculature or dissected trophoblast tissue (11, 28, 29, 30). Some studies have also provided evidence for calcium/calmodulin-independent iNOS activity in placenta (29, 31, 32, 33); however, other researchers did not find iNOS mRNA expression by RT-PCR of placental mRNA (34).

In attempt to investigate the interactions between ET-1 and the NO system in the feto-placental unit, we examined the mRNA expression of ET-1, iNOS, and eNOS in human cultured placental trophoblastic cells obtained from preeclamptic (PE cells) and normotensive (NT cells) pregnancies. We also studied whether exogenous ET-1 may affect the expression of iNOS and eNOS in human placental trophoblast cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation and culture of human placental trophoblastic cells

Placentas were obtained immediately after term delivery from six normal and seven preeclamptic nonlaboring pregnant women undergoing caesarian section at 35–40 and 30–36 weeks gestation, respectively. Informed consent was obtained from all patients. Preeclampsia was defined as a blood pressure of 140/90 mm Hg or higher or a rise of 15 mm Hg diastolic or 30 mm Hg systolic on two separate readings at least 6 h apart with proteinuria (>1+ or 300 mg/24 h) and edema. In the preeclamptic group, pharmacologically treated patients and diabetic or chronic hypertensive patients were excluded. All of the women were nulliparous. Clinical data from the two groups are given in Table 1Go.


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  		Table 1. Characteristics of normotensive (NT) and preeclamptic pregnant women

 
Trophoblast cells were isolated using a slight modification of the method of Nelson (35). The villous tissue was removed by sterile dissection from the centers of different cotyledons, excluding chorionic and basal plates, washed repeatedly with 0.9% sodium chloride to remove blood from the intervillous space, and minced with scalpel blades. Approximately 15 g whole tissue villi were placed into a 50-mL polypropylene tube and incubated with an equal volume of dispase (Becton Dickinson and Co., Bedford, MA) at 37 C for 15 min with constant shaking. The enzyme was inactivated by adding 200 µL 0.5 mol/L ethylendiamine tetraacetic acid, and the tissue suspension was centrifuged at 200 x g for 5 min at 4 C. The supernatant was discarded, DMEM (Life Technologies, Inc., Grand Island, NY) was added to the digest to the 50-mL mark, and the suspension was mixed and incubated on ice for 20 min, during which time the villous fragments settled to the bottom of the tube. The supernatant containing the trophoblast cells was transferred to another tube filtering through a 100-µm pore size nylon mesh. This procedure was repeated once. The two supernatants containing trophoblast cells were pooled and centrifuged at 200 x g for 10 min at 4 C. Further purification of the trophoblast cell pellet was performed by Percoll density gradient centrifugation (36). The gradients were made from 45% to 15% Percoll (vol/vol) in 5% steps of 1.5 ml each by dilutions of Percoll with Hank’s Calcium Magnesium Free and layered in a 15-mL conical polystyrene centrifuge tube. The cell pellet was resuspended in DMEM to a volume of 2 mL and then layered onto the Percoll gradients and centrifuged at 1850 x g for 30 min. Trophoblast cells were recovered with a Pasteur pipette in the density of the gradient between 1.084 and 1.062 g/mL. The cells were transferred to a fresh tube and centrifuged at 200 x g for 30 min at 4 C. The trophoblast cell pellet was washed with 25 mL DMEM and centrifuged again. The supernatant was aspirated and discarded. The cells were then resuspended in DMEM, counted on a hemocytometer, and checked for cell viability by trypan blue exclusion. The viability of the cells was greater than 90%. Using immunohistochemical staining techniques, approximately 95% of the cells stained for cytokeratin (a trophoblast marker). The cells were plated onto 96-mm culture dishes in DMEM and 10% FCS (10–40 x 106 cells/dish) and allowed to adhere for at least 15 h in humidified incubator gassed with containing 95% air and 5% CO2. The cells were cultured in serum-free DMEM with and without 10-7 mol/L ET-1 (Roche Molecular Biochemicals, Indianapolis, IN) for 12 and 24 h as indicated for each experiment.

RNA isolation and ET-1 Northern analysis

For the Northern analysis, total RNA was isolated from human cultured trophoblastic NT and PE cells by standard guanidinium isothiocyanate/CsCl gradient centrifugation and subjected to Northern blotting. Human ET-1 complementary DNA (cDNA) probe was obtained from the RT-PCR product. RT-PCR was performed from deoxyribonuclease-treated total RNA of trophoblastic cells followed by PCR using the Perkin-Elmer Corp. RNA Gene-Amp PCR Kit (Perkin-Elmer Corp./Cetus, Norwalk, CT). Amplification of ET-1 cDNA was performed with an annealing temperature of 60 C, using 5'-TTCCGTATGGACTTGGAAGC-3' and 5'-AAGCCAGTGAAGATGGTTGG-3' as 5'- and 3'-primers. PCR fragment was visualized by 2% agarose gel electrophoresis and ethidium bromide staining, then cloned using Original TA Cloning Kit (Invitrogen, San Diego, CA). The identity of RT-PCR-amplified cDNA was confirmed by nucleotide sequence analysis. This 32P-labeled ET-1 cDNA was used as a probe, and hybridization was carried out overnight at 42 C in hybridization buffer containing 50% formamide, 10% dextran sulfate, 1 mol/L NaCl, and 0.1 mg/mL denatured salmon sperm DNA. After hybridization, the blot was washed in 2 x SSC (standard saline citrate)-0.5% SDS at room temperature for 15 min and in 0.2 x SSC-0.5% SDS at 50 C for 15 min. The membrane was exposed to x-ray film for 12–24 h at -80 C. The filter was also hybridized with the probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control for RNA loading, and the bands were quantitated by scanning densitometry.

RT-PCR

mRNA was extracted from trophoblastic NT and PE cells cultured in medium alone or treated with ET-1 10-7 mol/L for 12 and 24 h. RT-PCR was performed by a first step RT from deoxyribonuclease-treated total RNA, followed by PCR. RT from 1 µg total RNA was conducted for 60 min at 37 C in 20 mL reaction cocktail containing 4 µl 5 x PCR buffer, 0.001 mol/L dithiothreitol, 10 mmol/L deoxy-NTP mix, 0.6 pmol/L oligo(deoxythymidine), 0.2 U ribonuclease inhibitor, and 10 U Moloney murine leukemia virus reverse transcriptase (200 U/mL; BRL, Gaithersburg, MD). The amount of starting material and the number of cycles were selected so that the amplified product signal was quantitatively related to the input of RNA. In detail, samples of the PCR reactions were taken at multiple points throughout the amplification, allowing analysis of the product during the exponential phase of DNA amplification for appropriate quantitation. Amplification of iNOS and eNOS cDNAs was performed at annealing temperatures of 56 and 62 C, respectively, using Taq DNA polymerase (Perkin-Elmer Corp./Cetus) and the following primers: iNOS(5'), 5'-GCCTCGCTCTGGAAAGA-3'; iNOS(3'), 5'-TCCATGCAGACAACCTT-3'; eNOS(5'), 5'-GACATTGAGAGCAAAGGGCTGC-3'; and eNOS(3'), 5'-CGGCTTGTCACCTCCTGG-3'.

PCR products were analyzed by 2% agarose gel electrophoresis followed by Southern blotting. The filters were hybridized with [{gamma}-32P]ATP end-labeled oligonucleotide probes that matched a portion of the iNOS and eNOS cDNA sequences, respectively, included in their amplified products. Analysis to quantify the bands was performed by scanning densitometry. Then, PCR products were cloned using the Original TA Cloning Kit (Invitrogen), and the identities of RT-PCR-amplified cDNAs were confirmed by nucleotide sequence analysis. GAPDH (sense and antisense primers: 5'-CTTCACCACCATGGAGGAGG-3' and 5'-TGAAGTCAGAGGAGACCACC-3') was used as an internal control for PCR.

Data analysis

Data are expressed as the mean ± SEM and were compared for significance using Student’s t test. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The mRNA expression of ET-1 was examined in human trophoblastic placental cells obtained from preeclamptic and normotensive pregnancies cultured for 24 h. Interestingly, we observed an increased ET-1 mRNA expression level in PE trophoblastic cells compared to NT trophoblastic cells. Figure 1Go (top) shows the Northern blot analysis from two different patients. NT and PE trophoblastic cells were cultured in medium alone or in the presence of ET-1 (10-7 mol/L) for 12 and 24 h. At 12 h, no effect was observed, whereas at 24 h, addition of exogenous ET-1 up-regulated ET-1 mRNA expression in both NT and PE trophoblastic cells compared to that in the same untreated cells (Fig. 1Go, bottom).



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  		Figure 1. Top, Northern blots showing an increase in ET-1 mRNA in PE trophoblastic cells compared with NT cells. Total cellular RNA was prepared from rat striatal slices. Five micrograms of total RNA were loaded on each lane. Blots were hybridized with 32P-labeled ET-1 cDNA. PE cells were trophoblastic cells obtained from preeclamptic pregnancies; NT cells were trophoblastic cells obtained from normal pregnancies; endothelial cells were the positive control. The bands were analyzed by scanning densitometry. Data are expressed as the mean ± SEM. *, P < 0.05. Bottom, Northern blots showing an increase in ET-1 mRNA in NT and PE trophoblast cells untreated (C) or treated (T) with ET-1 (10-7 mol/L). Data are expressed as the mean ± SEM. *, P < 0.05.

 
To investigate the possible interaction(s) between ET-1 and the NO system we first analyzed by semiquantitative PCR in the same cultured cells the mRNA expression of iNOS and eNOS basally and after ET-1 treatment. iNOS and eNOS mRNA expression was detected in both NT and PE trophoblastic cells. The expected product sizes were 499 and 425 bp, respectively. PE trophoblastic cells expressed lower iNOS mRNA levels than trophoblastic cells from normotensive pregnancies (Fig. 2AGo). On the contrary, eNOS mRNA expression was higher in PE trophoblastic cells than in NT cells (Fig. 2BGo). Moreover, in the presence of ET-1 we observed a decrease in iNOS (Fig. 2AGo) and an increase in eNOS (Fig. 2BGo) mRNA expression levels in both NT and PE trophoblastic cells compared with those in the respective untreated cells.



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  		Figure 2. Southern hybridization of RT-PCR-amplified iNOS and eNOS gene transcripts, GAPDH expression was used as an internal control of both RT and PCR. The results shown represent one point of the multiple samples taken during the exponential phase of DNA amplification for accurate quantitation, as described in Materials and Methods. The bands were analyzed by scanning densitometry. A, A lower level of iNOS expression was found in PE than in NT cells (C), and a decrease in the expression level occurred in both cells after ET-1 (10-7 mol/L) treatment (T). Data are expressed as the mean ± SEM. *, P < 0.05. B, A higher level of eNOS expression was found in PE than in NT cells (C), and an increase in the expression level occurred in both cells after ET-1 (10-7 mol/L) treatment (T). Data are expressed as the mean ± SEM. *, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The data of the present study characterize for the first time ET-1 and NOS mRNA expression and their regulation in trophoblastic cells from NT and PE placentas, suggesting an intriguing functional relationship between ET-1 and the NOS system. It is well known that high levels of ET-1 are observed in pregnant women with PE (8, 19, 20, 21, 37). The mechanism of ET increase in PE is unknown, but some hypotheses can be made. First, ET-1 is basically a local factor acting at the junction between the endothelium and the vascular smooth muscle layer; disruption and destruction of these anatomical boundaries can lead to a leak of ET from its local environment to the bloodstream, with subsequently higher peripheral blood levels. Second, an abnormal production of ET by the affected endothelium might be a primary mechanism for its increase, both locally and in the bloodstream. Third, increased production of ET-1 from placental or fetal tissue in PE or increased diffusion into the maternal circulation might explain the elevated levels found in PE.

In fact, ET-1 IR has been detected in trophoblast tissue and umbilical vessels of human placenta (38, 39, 40). A large amount of ET-1 is also present in amniotic fluid (41), and ET receptor is expressed in human fetal membranes, chorionic vessels, and decidua parietalis (14, 42, 43, 44); moreover, ET-1 mRNA is expressed in cultured trophoblast, fibroblast, and vascular smooth muscle cells from human placenta (16, 17, 45, 46). It must be pointed out that because of its frontier position in direct contact with maternal blood, the syncytiotrophoblast could have an endothelial-like function and, therefore, could be an important source of placental ET. The presence of high affinity binding sites for ET-1 on villous trophoblast and membranes (44) suggests an autocrine feedback pathway by which ET-1 regulates its own release.

In the present study we observed that trophoblastic ET-1 mRNA expression was higher in PE than in control subjects. Moreover, when ET-1 was added to the cultures, its own mRNA expression in both PE and NT cells was up-regulated. The possibility that such results could be partially ascribed to the different gestational ages of the two groups should be excluded, because Fant et al. (45) demonstrated that mRNA ET-1 expression in chorionic villous tissue is greater at term than in preterm placentas. Therefore, as PE placentas were collected at a younger gestational age than controls, the greater mRNA expression found in PE patients can be considered an intrinsic pattern of the ET-1 expression. Moreover, our data are further supported by the demonstration that ET-1 gene expression is increased in the placenta of preeclamptic gestations (47). However, conflicting results have been reported for ET levels in pregnant women with hypertension, which showed that placental ET-1 expression levels were comparable in placental tissue from preeclamptic and normal pregnant women and are significantly higher only in IUGR (48, 49). These differences may be due to the placental compartment analyzed, because Faxèn and Benigni used for analysis total RNA from placental fragments, including all components of placental tissue, such as endothelial cells and fibroblasts; on the contrary, we used only cultured trophoblast cells for RNA analysis.

Another important finding of the present study is the different mRNA expressions of iNOS and eNOS observed in PE and control cells. It is well known that NO is generated from the metabolism of arginine by NOS. It diffuses from endothelial cells to underlying smooth muscle, where it activates guanylate cyclase, increases GMP production, and leads to vascular relaxation. NOS expression and activity were demonstrated in both PE and normal placentas (28, 29, 33, 50, 51, 52). In the human feto-placental circulation, NO appears to attenuate the action of vasoconstrictor substances such as ET (27) and to contribute to the maintenance of low vascular resistance (26). Interestingly, infusion of donor NO into the maternal circulation of pregnancies reduces uterine artery resistance (53) and improves the umbilical flow velocity waveforms (54).

Myatt et al. (28) described the action of NO in the human feto-placental vasculature and mapped the distribution of type II and III isoforms of NOS. Biochemical characterizations showed that the NOS enzyme present in the feto-placental vasculature (31) or dissected trophoblast tissue (29) was the endothelial or type III isoform. Furthermore, immunohistochemical (11, 28, 30) and in situ hybridization (29) studies have shown that the type III isoform is localized to the endothelium of umbilical, chorionic plate, and stem villous vessels and also in the villous syncytiotrophoblast.

Several studies provided evidence for the presence, in the placenta, of a calcium-independent iNOS activity (31, 32, 33, 50), which represents the main source of the arginine metabolic pathway leading to NO synthesis (24); conversely, Garvey (34) and Schonfelder (55) did not find iNOS in normal placental tissue by RT-PCR. Moreover, Lyall et al. (51) were not able to demonstrate the expression of iNOS isoform in syncytiotrophoblast cell culture either basally or when exposed to cytokines.

There are several conflicting reports concerning this specific point. Imunostaining for type II NOS has been shown to be localized to stromal cells of the villous, and it was clearly distinct from the eNOS isoform immunostaining that was localized in the syncytiotrophoblast (32). In contrast, in our study iNOS expression was found in trophoblastic cell cultures. A hypothesis to explain these discrepancies could be the following. The time of culture before analysis may affect cell morphology; in fact, data by Lyall et al. (51) demonstrate that after 5 days, cultured cytotrophoblast cells from term placental tissue undergo differentiation into syncytiotrophoblast. In our study trophoblastic cells were cultured for 24 h before the analysis.

We also showed that basal eNOS mRNA expression was greater in PE than in control placental tissue, whereas iNOS expression was lower in trophoblast cells from PE placenta than in controls. Previously, no overt difference in eNOS immunostaining was seen in syncytiotrophoblasts from preeclamptic placenta compared with controls (52). Also, Conrad and Davis (50) found no difference in eNOS activity analyzed in villous trophoblast dissected from normo- and hypertensive placentas. In pregnancies complicated by preeclampsia and/or IUGR, Myatt et al. found an up-regulation of the type III NOS isoform in the altered vasculature of these placentas. Placentas from patients with PE with or without IUGR had a greater distribution of eNOS in syncytiotrophoblasts (30). Interestingly, the greater eNOS expression found in these pathological pregnancies may be an adaptive response to the increased vascular resistance and poor perfusion observed in their placentas.

In our experimental design, ET-1 increased the expression of eNOS and decreased the expression of iNOS in both PE and normal trophoblast cell cultures. The potential impact of these data is of paramount significance. In fact, ET is able to induce in normal trophoblast cells an expression pattern of inducible and endothelial NOS similar to that found in PE trophoblastic cells, suggesting that vasoconstrictor agents may work by inducing a significant change in the balance of NOS isoforms, which, in turn, may lead to an absolute or relative decrease in NO functional activity. Such a mechanism apparently also has the ability to be effective in pathological conditions, as those modifications induced by ET in NT cells were observed in PE cells. The functional relationship between ET and NOS isoforms seen in the present study suggests that PE induces a significant change in the balance of vasoactive substances in placental tissue.

In conclusion, we demonstrated significant modification of ET-1 and NOS isoform mRNA expression in pregnancies complicated by PE. ET-1 expression, as marker of endothelium damage and vasoconstrictor, is increased; on the other hand, iNOS expression, which represents the main source of NO, is decreased, and, thus, the amount of NO produced is inadequate to attenuate the vasoconstrictor action of ET-1. eNOS expression is increased, probably as an adaptive or compensatory mechanism. Furthermore, ET-1 is able to induce its own as well as eNOS expression and to reduce iNOS expression in cultured NT trophoblastic cells. This latter finding suggests the existence of functional relationships between ET(s) and NOS isoforms, which could constitute the biological mechanism leading to the reduced placental blood flow and increased resistance to flow in the feto-maternal circulation, which are characteristic of the pathophysiology of preeclampsia.

Received June 14, 1999.

Revised December 6, 1999.

Revised January 31, 2000.

Accepted February 27, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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