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Department of Obstetrics and Gynecology (M.I., A.I., M.N., S.M.) and Laboratory of Molecular Pathogenesis (T.S.), Nagoya University School of Medicine; and Maternity and Perinatal Care Center (Y.O.) and Division of Pathology, Clinical Laboratory (T.N.), Nagoya University Hospital, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan
Address all correspondence and requests for reprints to: Atsuo Itakura, M.D., Department of Obstetrics and Gynecology, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. E-mail: . aita{at}med.nagoya-u.ac.jp
Abstract
The purpose of this study was to elucidate the mechanisms underlying the regulation of feto-placental circulation mediated by the renin-angiotensin system under preeclamptic conditions. We measured angiotensin-converting enzyme (ACE) activity, protein expression, and mRNA expression in uncomplicated and preeclamptic placentas and examined the localization of ACE. In addition, ACE activity and mRNA expression in human umbilical venous endothelial cells (HUVECs) under hypoxic conditions were analyzed. ACE activity, protein expression, and mRNA expression in placental tissues from preeclampsia were all significantly higher than those from uncomplicated pregnancies. ACE activity in vessel fractions was extensively higher than that in trophoblast-rich or macrophage-rich fractions. Additionally, ACE activity in HUVECs was significantly higher than that in human arterial endothelial cells, and ACE mRNA was primarily localized to venous endothelial cells of stem villous in placentas. Furthermore, hypoxic condition induced both ACE activity and mRNA expression in HUVECs. These results suggested that venous endothelial cells within placental stem villous tissues and umbilicus play an important role in the regulation of the feto-placental renin-angiotensin system, and in response to hypoxic conditions the feto-placental unit seemed to induce ACE activity in the placenta; such an effect would be likely to lead to regulation of the fetal circulation.
PREECLAMPSIA IS characterized by maternal hypertension, proteinuria, and generalized edema. In addition to these effects on the mother, there is a profound effect on the fetus, frequently resulting in intrauterine growth retardation (IUGR) or perinatal death (1, 2); nevertheless, little is known about the cause. One of the main postulated mechanisms of preeclampsia is utero-placental hypoperfusion, in which the supply of both nutrients and oxygen to the placenta is disturbed. One of the causes may be a deficiency of the physiological remodeling of uterine spiral arteries (3, 4, 5). Without sufficient changes in the uterine vasculature in early pregnancy, the placenta may become hypoxic with advancing gestation and suffer from oxygen deficiency at the tissue level. Although numerous studies of preeclampsia were reported, the pathogenesis of preeclampsia remains obscure. Therefore, it is necessary to elucidate the pathogenesis or etiology of preeclampsia.
Angiotensin-converting enzyme (ACE; EC 3.4.15.1), a dipeptidyl carboxypeptidase, plays an important role in blood pressure homeostasis by generating angiotensin II (ANG II), a vasoconstrictor peptide also recently recognized as a growth factor, from angiotensin I (ANG I), the inactive peptide released after cleavage of angiotensinogen by renin. ACE, which also inactivates the vasodilator peptide bradykinin (6), is a membrane-bound enzyme located mainly on the luminal faces of endothelial cells in blood vessels (7). In plasma, ACE is present in a soluble form, originating mainly from endothelial cells by proteolytic cleavage (8). Our previous study demonstrated that the mean level of ANG I in umbilical venous plasma was lower than that in umbilical arterial plasma, whereas the opposite is the case for ANG II (9, 10). We also revealed ACE mRNA expression in placentas, which is possibly linked to the differences in ANG I and ANG II concentrations in the two sites. It is probable that the major site for conversion from ANG I to ANG II by ACE in the feto-placental unit is the placenta. It is also well known that ACE inhibitors are fetotoxic, possibly because of their effects on the feto-placental circulation. These data suggest an important role of ANG II (11).
On the other hand, whether the feto-placental renin-angiotensin system (RAS) in preeclampsia is accelerated is still controversial. Although Broughton-Pipkin and Symonds (12) previously showed that ANG II concentrations in maternal as well as umbilical plasma in preeclamptic patients were significantly higher than those in uncomplicated patients, some researchers reported no significant difference in maternal ACE activity in women with or without preeclampsia (13, 14, 15), and others found reduced maternal plasma ANG II levels in preeclampsia compared with those in normal pregnancy (16, 17, 18, 19).
Therefore, to assess whether feto-placental RAS is activated in preeclampsia, we first evaluated ACE activity and ACE expression in placentas with or without preeclampsia. Moreover, we examined ACE localization in placentas and, finally, ACE changes under hypoxic condition.
Materials and Methods
Source of clinical samples and preparation
Placental tissues were obtained at 34–41 wk gestation from uncomplicated and preeclamptic women delivered by cesarean section without labor. Uncomplicated women were normotensive and had no proteinuria or signs of preeclampsia before delivery. Although these included preterm premature rapture of membranes or cases with multiple pregnancies, those with clinical chorioamnionitis (fever, maternal or fetal tachycardia, uterine tenderness, foul odor, and leukocytosis) or women undergoing spontaneous preterm labor were excluded from this study. The preeclamptic group all met one or more of the following criteria: 1) systolic blood pressure above 160 mm Hg and/or diastolic blood pressure above 110 mm Hg on at least two occasions at least 6 h apart, and 2) proteinuria above 5 g/24 h or a dipstick reading of 3+ to 4+ in at least two random urine specimens collected at least 6 h apart. None of the cases of preeclamptic pregnancy had any other maternal complication. Gestational stages were determined by ultrasonographic examination in the first trimester.
Villous tissue blocks were taken from the decidual side of placentas, and small villous portions (
150–200 mg) were washed twice extensively in sterile ice-cold PBS, then frozen immediately in liquid nitrogen and stored at -80 C until use. Umbilical venous blood samples were obtained from the women as described above; these samples were taken at the placenta-umbilical cord junction immediately after clamping. The blood samples were centrifuged, and plasma was then frozen and stored -20 C until further examined.
The use of these tissues was approved by the ethics committee of Nagoya University School of Medicine, and written informed consent was obtained from each woman before clinical sampling.
Cell fractionation of placenta and placental cell culture
Placental tissues were obtained from women with uncomplicated pregnancies undergoing cesarean section at term with intact fetal membranes, and placental trophoblasts, macrophages, and vessels were isolated according to established procedures, essentially as described previously (20, 21). In brief, the villous tissues were removed by sterile dissection from the centers of different cotyledons, washed repeatedly with PBS, and minced with scalpel blades. Small pieces of placenta were transferred to buffered RPMI 1640 medium (Sigma, St. Louis, MO) containing 1 mg/ml collagenase (Wako Pure Chemical Co. Ltd., Osaka, Japan), 5 U/ml dispase (Godo Shusei, Tokyo, Japan), 30 U/ml deoxyribonuclease I (Sigma), 200 U/ml penicillin, 200 µg/ml streptomycin, and 5 µg/ml amphotericin and incubated at 37 C for 60 min with gentle stirring. The fragments of tissue were allowed to settle for 2 min, then the supernatant containing released cells was decanted and filtered through three layers of sterile gauze into sterile tubes. Vessel fractions obtained from the material remaining on the surgical gauze were minced with scalpel blades and placed into an ice-cold glass homogenizer containing 5 vol lysis buffer (ice-cold 50 mM Tris-HCl buffer with 0.25% Triton X, pH 7.4). After homogenization, the sample was centrifuged for 10 min at 3,000 x g at 4 C, and then the collected supernatant was centrifuged for 15 min at 10,000 x g at 4 C. The final supernatant was collected as the vessel fraction and stored at -80 C until use. For further purification, the supernatant containing placental cells was layered in 50-ml tubes containing a 30-ml 20–60% (vol/vol) Percoll (Sigma) gradient, in 10% steps of 6 ml each, by dilutions of Percoll with RPMI 1640 medium and centrifuged at 800 x g for 30 min at 4 C. Three bands of cells were isolated: one at density 1.033–1.048 g/ml (middle band), another at density 1.077–1.100 g/ml (bottom band), and a third at density greater than 1.140 g/ml containing red cells. The majority of cells in the middle band were trophoblasts, and more than 90% of them were stained for cytokeratin as a marker of trophoblast. In the bottom band the majority of cells were macrophages, and more than 95% of them were stained for CD68 as a marker of macrophage. Therefore, we called them the trophoblast-rich fraction and the macrophage-rich fraction, respectively. Each was transferred to a fresh tube, then was washed several times with 25 ml RPMI 1640 medium to eliminate Percoll and centrifuged at 1,850 x g for 30 min at 4 C. Freshly isolated cells of each fraction were seeded in culture dishes coated with collagen type I (Falcon) and incubated for 18 h at 37 C in humidified atmosphere of 21% O2 and 5% CO2 with 10% FCS. Finally, the medium was aspirated, the cells were washed with PBS twice, and then 300 µl lysis buffer were added. Cells were manually detached from the dishes with a cell scraper, lysed, and centrifuged for 15 min at 10,000 x g at 4 C, and the supernatant was stored at -80 C until use.
Culture of human umbilical venous endothelial cells (HUVECs) and human umbilical arterial endothelial cells (HUAECs)
Human umbilicuses were obtained from term placentas of uncomplicated women delivered by cesarean section. HUVECs and HUAECs were isolated according to methods described previously (22, 23). Cells were plated on collagen type I-coated dishes in Humedia-eg2 (Kuraubo Co., Tokyo, Japan) and incubated in a humidified atmosphere of 21% O2 and 5% CO2 at 37 C. After reaching confluence, cells were used within less than 72 h. Before use, each monolayer was inspected microscopically to ensure that only endothelial cells were present (identified by their typical cobble stone appearance). All experiments were performed with the same second to fifth passages. Incubator conditions were either normoxic (21% O2 and 5% CO2) or hypoxic (2% O2 and 5% CO2, balanced N2) in a humidified incubator with an interior temperature of 37 C. The medium (DMEM, Sigma) contained 5 mg/ml BSA (Roche Molecular Biochemicals, Barden, Switzerland) and was equilibrated to the environmental gas conditions overnight before cellular exposure. Reoxygenation was prevented through immediate replacement of hypoxic medium with lysis buffer while the cells were on ice.
Measurement of ACE activity
Preparation of placental homogenates was previously described (24). The sample of frozen placental tissue, weighing 150–200 mg, was minced into small pieces and immediately placed into an ice-cold glass homogenizer containing 5 vol lysis buffer. The suspended solution was well homogenized. Then the homogenized sample was centrifuged for 10 min at 3,000 x g at 4 C, and then the collected supernatant was centrifuged for 15 min at 10,000 x g at 4 C. The supernatant was employed for measurement of ACE activity. For HUVECs and HUAECs, 300 µl lysis buffer were added after aspiration of the medium and washing with PBS twice. Cells were manually detached from the dishes with a cell scraper, lysed, and finally centrifuged for 15 min at 10,000 x g at 4 C. The supernatant was collected for measurement of ACE activity.
ACE activities of plasma samples, HUVECs, HUAECs, placental fractions, and placental tissue extracts were measured by a colorimetric method using p-hydroxybenzoyl-glycyl-L-histidy-L-leucine as the substrate (25). The concentration of quinoneimine dye was quantitated at its absorbance maximum at 505 nm. The specificity of the assay for ACE activity was confirmed by the absence of colorimetric signals when 10-6 M captoril was added to the initial incubation of substrate with sample. The protein concentration of each tissue extract was measured with the bicinchoninic acid protein assay kit (Pierce Chemical Co., Rockford, IL).
Western blot analysis
Western blotting was carried out according to a method previously described (26). Aliquots of 10 µg protein extract were separated on 10% SDS-polyacrylamide gel under reducing conditions, followed by electrophoretic transfer onto nitrocellulose membranes (Millipore Corp., Bedford, MA). The membranes were blocked in 5% nonfat milk prepared in PBS containing 0.05% Tween 20, then immunoblotted with monoclonal anti-ACE antibody (1:100; Chemicon International, Inc., Temecula, CA). The membranes were washed with PBS containing 0.05% Tween 20 and incubated with horseradish peroxidase-conjugated antimouse IgG polyclonal antibody (1:1,000; Promega Corp., Madison, WI). After washing the membranes, the blots were detected by an enhanced chemiluminescence method using an ECL Plus Western blotting detection kit (Amersham Pharmacia Biotech, Piscataway, NJ). The results were visualized by fluorography using RX-U medical x-ray film (Fuji Photo Film Co., Ltd., Kanazawa, Japan). Anti-ß-actin monoclonal antibody (1:5,000; Sigma), its second antibody, as described above (1:10,000), was applied as a control. For quantitation of protein expression, a scanning densitometer (model GS-700, Bio-Rad Laboratories, Inc., Richmond, CA) was used, coupled with personal computer analysis software (Bio-Rad Laboratories, Inc.).
In situ hybridization
For in situ hybridization, placental tissues from uncomplicated and preeclamptic women (34–41 wk gestation) delivered by cesarean section were immediately fixed as follows. Placentas were manually dissected from other tissues, and samples were cut into 0.5- to 1.0-cm3 portions, fixed in 4% paraformaldehyde dissolved in PBS, embedded in paraffin, and cut serially at 3-µm thickness.
Antisense and sense RNA probes complementary to bases 3919–3990 and 690–818 of human ACE were synthesized. The plasmid vector (pBluescript KS) containing approximately 3.9 kb coding region and 0.1 kb 3'-untranslated sequence (27) was cut with Eco81I (Perkin-Elmer Corp., Foster City, CA) to yield 3.2-, 2.4-, and 0.6-kb fragments, and the first of these was isolated. This linearized plasmid contained RNA polymerase promoter sites (T7 and T3). Transcription of biotin-labeled RNA probes was performed with T3 RNA polymerase to synthesize the antisense probe and with T7 RNA polymerase to synthesize the sense probe, using biotin-14-CTP (Life Technologies, Inc., Gaithersburg, MD) and Riboprobe In Vitro Transcription Systems (Promega Corp.).
Tissue sections were deparaffinized using a traditional method. In situ hybridization was performed with an In Situ Hybridization Detection System for Biotinylated Probes (DAKO Corp., Carpinteria, CA) according to the manufacturer’s protocol. The site of hybridization results in the deposition of an insoluble blue-purple product. Finally, the sections were counterstained using Nuclear Fast Red counterstain (Biomeda Co., Foster City, CA) before mounting.
RNA isolation and Northern blot analysis
Total RNA was isolated from HUVECs and placental tissues using an RNeasy kit (QIAGEN, Tokyo, Japan) and stored at -80 C until use. Aliquots of 20 µg total RNA were lyophilized, denatured, and electrophoresed through 1.0% agarose gel containing 20 mM 3-(N-Morpholino)propanesulfonic acid, 5 mM sodium acetate, 1 mM EDTA (pH7.0), 0.66 M formaldehyde, and 2 µg/ml ethidium bromide, as described previously (28), then transferred to Hybond-N+ nylon membranes (Amersham Pharmacia Biotech, Arlington Heights, IL) using a vacuum blotting system and cross-linked to the membrane by UV irradiation. The efficacy of the transfer was confirmed by ethidium bromide staining, which was examined before and after transfer. The plasmid vector described above was cut with EcoRI and BglII (Perkin-Elmer Corp.) to yield 1.7- and 1.6-kb inserts of ACE cDNA. Both fragments were separated from the 3.0-kb vector, labeled with [32P]dCTP using the multiprime DNA labeling system (Amersham Pharmacia Biotech), and used as probes for ACE mRNA. ß-Actin cDNA, a housekeeping gene, was applied as a control. Hybridization was performed at 68 C for 2 h with gentle agitation in Perfect Hyb hybridization solution (Toyobo Ltd., Osaka, Japan). The membranes were washed according to the manufacturer’s protocol, and then autoradiographed. Each was first hybridized with the ACE probe, then reprobed and rehybridized with the ß-actin probe. The mRNA levels were calculated after normalization to ß-actin expression on the basis of the hybridization signals, as measured with a BAS 2000 Bioimage Analyzer (Fuji Photo Film Co., Ltd.).
Statistical analysis
The data presented are the mean ± SD. Statistical analysis was performed using the StatView program (version 4.5, Abacus Concepts, Inc., Berkeley, CA). The Mann-Whitney U test was used to estimate differences between uncomplicated and preeclamptic groups for ACE activity, ACE protein, and ACE mRNA expression in placental tissues and umbilical venous plasma samples. Similarly, Mann-Whitney U test was used to estimate differences in ACE activity in HUVECs compared with HUAECs. The statistical analysis used to test the significance of changes in ACE activity in placental fractions, kinetic analysis, and ACE mRNA induction in HUVECs was one-way factorial ANOVA, followed by Fisher’s protected least significant differences test. P < 0.05 was considered statistically significant.
Results
ACE activity in placental tissue extracts and umbilical venous plasma samples
To determine whether ACE activity changes in preeclampsia, ACE activity in placental tissue extracts and umbilical venous plasma samples was measured. Clinical characteristics of the cases are shown in Table 1
. There were no significant differences, except regarding mean birth weight, which was significantly lower in the preeclamptic cases (P < 0.01). ACE activity in the placentas in the preeclamptic group was significantly higher than that in the uncomplicated group (Fig. 1A). The mean value of ACE activity in preeclampsia was significantly higher: 6.59 ± 2.48 vs. 4.17 ± 1.46 U/G protein-coupled receptor (mean ± SD; P < 0.01). Similarly, the mean value of ACE activity in umbilical venous plasma samples was significantly higher in the preeclamptic samples compared with that in the uncomplicated samples: 13.61 ± 5.15 vs. 9.43 ± 2.75 U/liter (mean ± SD; P < 0.05; Fig. 1B).
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Next we examined whether ACE protein and mRNA expression changes in preeclampsia. Western blot analysis was performed using a mouse anti-ACE antibody to detect ACE protein expression in placental tissue extracts. ACE protein was detected as a single band with a molecular mass of approximately 170 kDa (Fig. 2A). As shown in Fig. 2B, ACE protein expression in preeclamptic placentas was 3.0-fold higher than that in uncomplicated placentas (P < 0.05). On the other hand, we could detect two endothelial ACE mRNAs of different size in isolated RNA by Northern blot analysis, one a 4.3-kb band of somatic form, the other a 3.5-kb band, which could not be quantified because of a faint visible band. Figure 2C compares the expression of ACE mRNA levels in placentas from the uncomplicated group with those from the preeclamptic group. As shown in Fig. 2D, the mean ACE mRNA level of preeclamptic placentas corrected by ß-actin mRNA was 1.5-fold higher than that of uncomplicated placentas (P < 0.05).
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To elucidate in which fraction of the placenta and the umbilicus ACE activity was predominant, ACE activity of trophoblast-rich fractions, macrophage-rich fractions, and vessel fractions was measured using isolated lysates from uncomplicated placentas. Also, ACE activity in HUAECs and HUVECs isolated from uncomplicated umbilicus was measured. Figure 3 shows that ACE activity in vessel fractions was the highest among them (P < 0.01); the mean values of vessel fractions, trophoblast-rich fractions, macrophage-rich fractions, and total placentas were 14.34 ± 2.44, 2.73 ± 1.03, 1.78 ± 0.75, and 4.15 ± 1.46 U/G protein-coupled receptor, respectively. The distribution of ACE activity in preeclamptic placenta was similar to that in uncomplicated placenta (data not shown). On the other hand, the mean ACE activity was significantly higher in HUVECs than in HUAECs: 46.07 ± 10.81 U vs. 4.54 ± 1.13 U/G protein-coupled receptor (mean ± SD; P < 0.01; data not shown).
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Figure 4A shows a tissue section hybridized with the antisense ACE probe. The signals in preeclamptic placentas were primarily localized in venous endothelial cells of stem villous tissue. Hybridization signals of ACE mRNA were also weakly detected in trophoblasts and macrophages. In the case of uncomplicated placentas, the ACE signals were similarly detected in venous endothelial cells, but were extremely weak (data not shown). Figure 4B shows control tissue hybridized with the sense probe of ACE.
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To evaluate whether ACE activity and ACE mRNA expression were induced in response to hypoxia, ACE changes in HUVECs under hypoxic conditions (2% O2) were assayed. Figure 5A shows that the hypoxic condition significantly induced ACE activity in the cell lysates within 2 h. ACE activity was increased in the time-dependent manner within 12 h (5.6-fold at peak). On the other hand, Fig. 5B shows the time course of ACE mRNA expression by Northern blot analysis. Similarly, hypoxia induced ACE mRNA expression within 2 h and peaked at 4 h, to 1.8-fold of the control value. The significant increase in ACE mRNA expression continued until 12 h (Fig. 5C).
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In the current study we revealed that ACE activity in placental tissues as well as umbilical venous plasma samples was significantly higher in preeclampsia than in uncomplicated pregnancies. We also demonstrated that both ACE protein and ACE mRNA were more strongly expressed in preeclamptic placentas compared with those in uncomplicated placentas. To our knowledge this is the first report on the activation of ACE in preeclampsia, whereas Broughton-Pipkin, Symonds, and our previous study (10, 12, 29) showed that the level of ANG II in umbilical venous plasma was higher than that in umbilical arterial plasma in both groups, and the level of ANG II in maternal peripheral venous plasma was much higher in preeclampsia than in uncomplicated pregnancy. The level of ANG II in umbilical venous plasma in preeclampsia was also much higher than that in uncomplicated pregnancy, with its steeper gradient between umbilical venous and arterial plasma (12). Therefore, their data suggested that ACE activity in preeclamptic placenta would be elevated. On the contrary, Kalenga (30) reported that no significant difference was found in the levels of ACE and ANG II in placenta between uncomplicated pregnancy and preeclampsia. This observation is in disagreement with our present study; however, it could be explained by the possibility that the differences in the investigation methods used could affect the results. They analyzed the levels of ACE and ANG II in placentas obtained from uncomplicated and preeclamptic women delivered vaginally; in addition, their data included only moderate preeclampsia without IUGR in preeclamptic groups. In contrast, all placental and umbilical samples in our experiments were obtained from women delivered by cesarean section in both groups, and we included only severe preeclampsia with IUGR because we previously demonstrated that ANG II concentrations in both umbilical vein and artery at vaginal delivery of uncomplicated women were higher than those at cesarean delivery without labor of uncomplicated women (31), and some researchers suggested changes in the feto-placental RAS in the advanced stage of preeclampsia with IUGR (12, 32, 33). In addition, it has been shown that ANG II levels in the fetus rise during the second stage of labor, especially during a difficult and prolonged delivery (12). Therefore, it seems that another factor or stress concerning vaginal delivery also induces placental ACE activity, which results in higher content of ANG II in umbilical vein even in the case not suffered preeclampsia.
Turning now to localization of ACE, our study directly revealed that ACE mRNA localized primarily in vessel fraction of the placenta, especially in venous endothelial cells of stem villous of the placenta. ACE activity in vessel fractions was significantly higher than that in other fractions. Moreover, ACE activity of HUVECs was significantly higher than that of HUAECs. This evidence is consistent with that previously reported. Caldwell first reported ACE localized in vascular endothelial cells (34), and Defendini (35) showed ACE activity in placental homogenates, including endothelial cells of feto-placental vasculature and syncytiotrophoblastic membrane. Consequently, we speculated that the placental and umbilical venous endothelial cells would be the main site for the conversion of ANG I to ANG II to regulate fetal circulation, and feto-placental circulation might serve as a substitution for pulmonary circulation in the adult or child RAS.
A series of investigations of physiological regulation of feto-placental circulation have been conducted in sheep (36, 37, 38, 39, 40). The ability of the fetus to redistribute its blood flow appropriately in response to reduced oxygen supply is of great importance in its survival and development. It is well known that the fetal plasma ANG II level rises during hypoxic episodes in sheep. Although to date the role of ANG II in cardiovascular control under hypoxia has not been fully elucidated even in sheep, ANG II is an alternative candidate for regulatory factor of this redistribution in the fetus. On the other hand, Kingdom (41) investigated the relation between umbilical arterial oxygen partial pressure and ANG II levels in the umbilical vein during human delivery. They found that when the human fetus suffered from low oxygen levels caused by stress, the level of ANG II in the umbilical vein was rising. This indicates that, as in the sheep fetus, under hypoxic conditions the ANG II level increases in the human fetus. A major finding in this study was that the hypoxic condition induced ACE mRNA as well as its activity in HUVECs. In preeclamptic women, fetus, placenta, and umbilicus are exposed in chronic hypoxic conditions. When HUVECs cultured under hypoxic conditions represent the other feto-placental vascular beds in the hypoxic condition, we thus speculate that in response to hypoxic stress, venous endothelial cells of placental stem villous produce ACE and stimulate the conversion from ANG I to ANG II, then feto-placenta blood flow will be redistributed.
In conclusion, we clarified that ACE was primarily localized in venous endothelial cells of stem villous tissue in the placenta, and ACE activity, ACE protein expression, and ACE mRNA expression were higher in preeclamptic placenta than in placenta from uncomplicated pregnancy. We also revealed that the hypoxic condition induced ACE mRNA expression as well as its activity in HUVECs in vitro. Therefore, it follows from our data that venous endothelial cells within placental tissue and umbilicus play an important role in the regulation of feto-placental RAS, and in response to hypoxic conditions, such as preeclampsia, the fetoplacental unit induces ACE activity in the placenta. Such an effect is likely to lead to regulation of the fetal circulation. Further studies are required to clarify the importance of the role of the feto-placental RAS. Such studies will bring further understanding to the pathogenesis of preeclampsia.
Acknowledgments
We are grateful to Dr. Michinari Hamaguchi (Laboratory of Molecular Pathogenesis, Nagoya University School of Medicine) for valuable technical advice, and to Ms. Mariko Ohkawa for excellent typing of this manuscript.
Footnotes
This work was supported by Grant-in-Aids from the Ministry of Post and Telecommunications of Japan for specific medical research (collaboration with Nagoya Teishin Hospital) and by Ogyaa donations.
Abbreviations: ACE, Angiotensin-converting enzyme; ANG, angiotensin; HUAEC, human arterial endothelial cell; HUVEC, human umbilical venous endothelial cell; IUGR, intrauterine growth retardation; RAS, renin-angiotensin system.
Received August 27, 2001.
Accepted January 9, 2002.
References
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, Uncomplicated pregnancy; •, preeclampsia. B, ACE activity in umbilical venous plasma samples. Columns and vertical bars represent the mean ± SD of 16 samples from each group. *, P < 0.05 (vs. uncomplicated).
