Differential Roles of Renin and Angiotensinogen in the Feto-Maternal Interface in the Development of Complications of Pregnancy
Eriko Takimoto-Ohnishi,
Tomoko Saito,
Junji Ishida,
Junji Ohnishi,
Fumihiro Sugiyama,
Ken-Ichi Yagami and
Akiyoshi Fukamizu
Tsukuba Advanced Research Alliance (TARA) (E.T.-O., T.S., J.I., A.F.), and Institute of Basic Medical Sciences, Laboratory Animal Resource Center (F.S., K.Y.), University of Tsukuba, Tsukuba, Ibaraki 305-8577; and Division of Biological Sciences (E.T.-O., J.O.), Graduate School of Science, Hokkaido University, Sapporo 060-0810; and Department of Molecular Cell Biology (E.T.-O., J.O.), Medical Research Institute, Tokyo Medical & Dental University, Chiyoda-ku, Tokyo 101-0062, Japan
Address all correspondence and requests for reprints to: Dr. Akiyoshi Fukamizu, Center for Tsukuba Advanced Research Alliance, Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan. E-mail: akif{at}tara.tsukuba.ac.jp.
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ABSTRACT
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We previously identified a transgenic mouse model that developed pregnancy-associated hypertension (PAH) and intrauterine growth restriction (IUGR) by mating females expressing human angiotensinogen (hANG) with males expressing human renin (hRN). These phenotypic defects were not observed in the opposite type of mating combination, despite the feto-placental overexpression of hRN and hANG detected in both types of crossbreeding. Detailed analysis of transgene localization in the labyrinth and its permeability to the maternal circulation revealed that hRN produced in trophoblast giant cells was secreted into the maternal circulation, whereas hANG, produced in chorionic trophoblasts and trophoblastic epithelium, was undetectable in the maternal plasma, probably due to their distinct spatial and temporal expression in labyrinth. These results demonstrated that PAH and IUGR could be mediated by feto-placental hRN through its permeability to the maternal circulation, not by feto-placental hANG production. Furthermore, overexpression of maternally derived hANG in decidua and spiral arteries of pregnant females with PAH and IUGR raises the possibility of local activation of the renin-angiotensin system and its pathophysiological effects on placental hypoperfusion in complications of pregnancy. This study provides in vivo evidence that the cell-specific expression of RN and ANG in the feto-maternal interface impacts their differential roles in pregnancy.
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INTRODUCTION
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THE PRIMARY FUNCTION of the placenta is to act as an interface between mother and fetus that allows, and even promotes, fetal growth and development, and to contribute to the maternal cardiovascular adaptations of pregnancy. Appropriate perfusion of the placenta is necessary for its critical endocrine and exchange functions. Normally, the physiological remodeling of spiral arteries reduces maternal blood flow resistance and increases uteroplacental perfusion to meet the requirements of the growing fetus (1, 2). Disturbance of the normal circulatory adaptation is a core predictor of abnormal pregnancy (3, 4, 5) but, despite numerous research efforts, the molecular basis underlying the regulation of local blood flow and vasculature in the feto-maternal interface remains unclear.
One of the postulated mechanisms by which the placenta may influence maternal vascular tone is the placental production of renin (RN) (6, 7), an aspartyl proteinase that cleaves angiotensinogen (ANG) to generate the decapeptide angiotensin I (AI). AI is further processed by angiotensin-converting enzyme to the potent vasoconstrictor peptide angiotensin II (AII). This effector molecule of the renin-angiotensin system (RAS) exhibits its bioactivities via different types of its specific receptor, AT1 and AT2 (8). All the components of the RAS have previously been shown to be present in the placenta (7, 9) and in and around the decidual spiral arteries (9, 10, 11). A growing body of evidence supports the existence of a local intrinsically active RAS in these tissues, which appears to participate in the regulation of uteroplacental blood flow and decidual vascular remodeling during pregnancy. Several indirect lines of evidence indicate that the up-regulation of RAS in the placenta might be important for the pathogenesis of some clinical disorders, such as pregnancy-associated hypertension (PAH) and preeclampsia (12, 13, 14). In addition, recent genetic studies have suggested that elevated expression of maternal ANG in human decidual spiral arteries may promote impaired remodeling of these vessels and reduce uteroplacental blood flow, potentially initiating events for preeclampsia (15, 16) and for intrauterine growth restriction (IUGR) (17). All these data indicate that locally activated RAS components in the feto-maternal interface could play a crucial role in the pathogenesis of complications of pregnancy.
Our previous study in an animal model, which mated transgenic mice expressing human RAS components, presented direct in vivo evidence that paternally derived human renin (hRN) produced in the placenta is secreted into the maternal circulation and regulates maternal blood pressure (18). In this model, transgenic females expressing human ANG (hANG) developed hypertension in late pregnancy, only when they had been mated with transgenic males expressing hRN. Recently, Bohlender et al. (19) reported an analogous model for the rat with identical transgene overexpression. These studies do confirm the utility of this transgenic animal model for an understanding the pathogenesis of hypertension in pregnancy. Our transgenic mice model with gestational hypertension also exhibited placental ischemic changes including necrosis and edematous enlargement (18, 20). In addition, their fetuses displayed growth restriction in size and weight (20). Our findings in this model make it clear that PAH and IUGR could be induced by the combined action of placental RN and maternal ANG. In the current study, we showed that these phenotypic defects of the mother and fetuses were not developed in the opposite type of mating combination, despite the overexpression of hRN and hANG detected in the fetal placenta of both types of crossbreeding. To address the discrepant phenotypes, we elaborated the cell types that express hRN and hANG in the placenta by in situ hybridization. Our detailed analysis provides the first in vivo evidence that the cell type-specific expression of RN and ANG in the feto-maternal interface impacts their differential roles in pregnancy.
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RESULTS
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Different Phenotypes in the Mother and Neonate of Transgenic Pregnant Mice
We previously showed that transgenic pregnant mice expressing hANG, when mated with transgenic males expressing hRN, displayed a transient elevation of blood pressure in late pregnancy. The blood pressure returned to normal levels after delivery of the pups (18). This induction of hypertension was correlated with an elevation of maternal circulating AII levels. Indeed, administration of an AII antagonist was effective in reducing the blood pressure in this model (pregnancy-associated hypertensive mice, PAH mice) (20). The PAH mice also exhibited placental ischemic changes including necrosis and edematous enlargement associated with fetal growth restriction (18, 20). During the cross-mating experiments, we observed that transgenic pregnant mice expressing hRN, when mated with the hANG males, displayed normal blood pressure throughout pregnancy and normal levels of circulating AII compared with those observed for wild-type (WT) pregnant mice (Fig. 1A
and Table 1
). In the nonhypertensive transgenic pregnant mice (non-PAH mice), the neonatal weights and sizes were indistinguishable from those of WT mice, and the histopathological analysis of placenta in non-PAH mice revealed no observable differences with WT placenta at 19 d of gestation (Fig. 1B
and Table 1
).

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Fig. 1. Legend on next page.
Phenotypes Observed in the Mother and Neonate during the Crossbreeding Transgenic Mice
A, Changes in systolic blood pressure of pregnant mice. Mating combinations are shown above left. Results are expressed as mean ± SDs for each determination. d 0 is the day of coitus; d 20 was the day of delivery, and systolic blood pressure was measured afterward. n, Total number of mice. B, Comparison of neonatal growth and placental morphology. Neonates (0 day) derived from the PAH mice, the nonhypertensive transgenic pregnant (non-PAH) mice, and WT control pregnant mice are shown (top). Their placentas at 19 d of gestation were stained histochemically with hematoxylin and eosin according to standard procedures. Bottom panels show high magnification views of the labyrinth. Arrows indicate the edematous enlargement characterized by the dilation of trophoblast in the layer of labyrinth. Magnifications, x1 (top); x20 (middle); x80 (bottom).
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Table 1. Comparison of parameters between mice with pregnancy-associated hypertension and nonhypertensive pregnant mice
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Expression of hRN and hANG in the Feto-Maternal Interface
In both types of crossbreeding between mice bearing human transgenes, the fetus and fetal-side of placenta (fetal placenta) were genotyped for both hRN and hANG. Northern blot analysis showed the tissue-specific expression pattern of transgenes at 19 d of gestation (Fig. 2A
). The existence of local RN and ANG synthesis in the placental tissues is consistent with previous data in humans (7, 9, 10, 11). The first point to note is that the overexpression of hRN and hANG were observed in the fetal placenta of both types of crossbreeding (Fig. 2
). Equivalent results regarding mRNA expression of both transgenes in placenta were shown at an analogous model for the rat (19). In this study, protein levels of active and inactive hRN were similar between the two lines (Fig. 2B
), consistent with the results of Northern blot analysis (Fig. 2A
). By contrast, hANG protein levels of PAH mice were 2.9-fold higher than those observed in the non-PAH mice (P < 0.005 by Students t test) (Fig. 2C
), even though the same levels of mRNA were observed in both the pregnant mice (Fig. 2A
). These results indicate that hANG production in the fetal placenta of PAH mice may be regulated at the translational level, but its precise mechanisms are not clear. Next in significance is that hANG mRNA and its proteins in the maternal decidua of PAH mice were highly expressed (Fig. 2
, A and C). We also detected overexpression of hRN and hANG in the fetal kidney and liver, respectively (Fig. 2A
), as well as in the fetal placenta; however, regardless of these results, the mother and neonate did not display any obvious abnormalities when the hRN females were mated with the hANG males (Fig. 1
and Table 1
).

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Fig. 2. Expression of hRN and hANG at 19 d of Gestation
A, Northern blot analysis. RNA samples from fetal placenta (fp) and decidua (d) of the PAH, non-PAH, and WT pregnant mice, and fetal kidney (k), liver (l), and maternal kidney of the non-PAH mice, and maternal liver of the PAH mice were subjected to Northern blot analysis with probes for hRN and hANG. Ethidium bromide (Etbr) staining of the gel is shown for comparison of applied amounts of RNA. B, Active and inactive hRN concentrations in the tissue extracts of fetal placenta were measured by RIA. C, hANG concentrations in the tissue extracts of fetal placenta and decidua were measured by RIA. Six animals were used for each determination in panels B and C. Values are expressed as mean ± SD. N.D., Not detectable.
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Local Activation of the RAS in the Feto-Maternal Interface
To clarify the cause of contrasting phenotypes observed in the mother and fetus between the two mating combinations, we first examined a local activation of RAS in the fetal placenta and in the maternal decidua by measuring the tissue levels of AI using RIA at 19 d of gestation. Whereas elevation of AI contents were detected in the fetal placenta of both PAH and non-PAH mice, compared with WT pregnant mice (Fig. 3A
), at the maternal decidua, only the PAH mice displayed an increase of AI production (Fig. 3B
). These results could provide the possibility that an activation of local RAS in the maternal decidua, not in the fetal placenta, may also trigger the development of complications in our transgenic pregnant mice.

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Fig. 3. Local Activation of the RAS in the Feto-Maternal Interface
AI levels in the tissue extracts of fetal placenta (A) and maternal dedcidua (B) at 19 d of gestation were measured by RIA. The lines of pregnant mice are indicated below. Four to six animals were used for each determination, and values are the means and SDs. Statistical evaluation of difference between groups of pregnant mice was done with the Students t test.
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Temporal and Spatial Characteristics of hRN and hANG Gene Expression in the Placenta
To elucidate the mechanisms underlying the regulation of local RAS activation in the placenta, we next examined transgene localization by in situ hybridization at different gestational ages. First, we found that fetal-derived hRN and hANG genes were expressed in distinct temporal and spatial patterns at the labyrinthine layer of both PAH and non-PAH mice. hANG mRNA was expressed in chorionic trophoblast cells and trophoblastic epithelium at d 11 (Fig. 4
, H and P), and its expression level was elevated at d 13 and markedly decreased at the end of gestation (Fig. 5
). At 15 d of gestation, the fetal hANG expression in labyrinth of PAH mice exhibited lower levels compared with that of non-PAH mice (Fig. 5A
). This result may imply the down-regulated expression of hANG gene in the labyrinth of PAH mice at d 15, but needs further investigation. hANG mRNA was also localized to the endoderm of visceral yolk sac (Figs. 4O
and 6F
) and to the endothelial cells of fetal vessels (Fig. 6
, FH). By contrast, the fetal-derived hRN gene was expressed only in trophoblast giant cells of the labyrinth and was first detectable at d 13 and gradually increased throughout gestation (Fig. 5
). This expression pattern of hRN in the labyrinth was quite similar to that of placental lactogen (PL)2 (21).

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Fig. 4. Localization of hRN and hANG mRNA at 11 d of Gestation in the Placental Tissues from PAH (AL) and non-PAH (MP) Mice
Trophoblast giant cells were identified by in situ hybridization for PL1 (C) and PL2 (D). Maternally derived hANG was widely expressed in the decidua (A). Its strong staining could be seen in cells lying close to the trophoblast giant cells (E) and adjacently to the endothelial cells of maternal blood sinuses (red arrow in A and F). At maternal vessels close to the circular smooth muscle layer, hANG signal was also detected in surrounding cells (red arrowhead in A and G). The fetal-derived hANG gene was expressed in chorionic trophoblast cells (white arrowheads in H and P) and trophoblastic epithelium (white arrows in H and P) in the surface of the labyrinth of both types of crossbreeding. Serial sections were analyzed by hybridization for MT1-MMP to identify the endothelial cells of maternal blood sinuses (J) and vessels (K). Weak signal of MT1-MMP was detected in chorionic trophoblast cells in the labyrinth (white arrowhead in L). In addition, cells at the junction between the invasive trophoblast giant cells and the maternal decidual cells contained mRNA of MT1-MMP (red arrowheads in I). Expression of the fetal origin hANG gene was also detected in the endoderm of visceral yolk sac of non-PAH mice (O), as well as of PAH mice (data not shown). Maternally derived hRN was expressed in endometrial epithelium (black arrow in M), and its weak signal was detected in the endothelial cells of maternal blood sinuses of decidua (black arrowhead in M and N). No signal of fetal-derived hRN was detected in this stage (data not shown). de, Decidua; la, labyrinth; my, myometrium; mv, maternal vessel; sp, spongiotrophoblast; tg, trophoblast giant cell. Magnifications, x10 (AD); x40 (M); x100 (N and O); x160 (E and I); x200 (FH, JL, and P).
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Fig. 5. Distinct Temporal and Spatial Expression of hRN and hANG mRNAs in the Placenta during Pregnancy
A, Distinct expression patterns of hRN and hANG mRNAs were seen in the layer of labyrinth of both PAH and non-PAH mice. de, Decidua; la, labyrinth; sp, spongiotrophoblast. Magnification, x20. B, The labyrinthine layer of non-PAH mice is shown at higher magnification (x200). Serial sections were stained with hematoxylin and eosin (HE). Red arrows, Trophoblast giant cells; white arrowhead, chorionic trophoblast cells; white arrow, trophoblastic epithelium.
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Fig. 6. Expression of hANG mRNA in the Maternal and Fetal Vessels at d 15 (AG) and at d 19 (H)
The maternal vessels of PAH mice (AE) and the fetal vessels of non-PAH mice (FH) were shown. Strong signal of hANG was detected in decidual cells surrounding the maternal blood sinuses (red arrowhead in A and D). Both MT1-MMP and PL2 mRNAs were expressed in the trophoblast-lined blood spaces located within spongiotrophoblast layer (black arrows in B and C), where hANG signal was not detected. At the maternal large arteries located in myometrium, hANG mRNA was detected in smooth muscle cells (red arrow in E), as well as in stromal cells. hANG mRNA was expressed in the endothelial cells of fetal vessels (white arrows) of visceral yolk sac (F) and labyrinth (G). hANG signal could also be seen in stromal cells of the umbilical cord (white arrowhead), and in endothelial cells of the umbilical arteries (white arrow), not of the veins (H). Its serial section was analyzed by Elastica Masson stain (I). hANG expression in these fetal vessels was also detected in the PAH mice (data not shown). Magnifications, x25 (AC); x80 (H and I); x100 (D, F, and G); x200 (E).
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Next, we found that maternally derived hANG was widely expressed in the decidua of PAH mice at d 11, and its high level expression was sustained until term. hANG mRNA was predominantly localized to cells lying in close to the invasive trophoblast giant cells (Fig. 4E
), cells lying adjacently to the endothelial cells of maternal blood sinuses (Figs. 4F
and 6D
), and cells surrounding the maternal dilated vessels from spiral arteries (Fig. 4G
). hANG expression was also detected in surrounding stromal cells and smooth muscle cells of large arteries located in the myometrium (Fig. 6E
). Using serial sections, we examined the localization of membrane type matrix metalloproteinase-1 (MT1-MMP), which may function in the tissue remodelling and cell invasion processes that take place during implantation and placentation (22, 23). Interestingly, adjacent localizations of hANG and MT1-MMP mRNAs could be detected in the decidua at 11 d of gestation (Fig. 4
).
In the non-PAH mice, we detected maternally derived hRN in the endometrial epithelium and in the endothelial cells of decidual blood sinuses at 11 d of gestation (Fig. 4
, M and N). hRN expression in the endometrial epithelium was continued until term, whereas hRN in the endothelial cells of blood sinuses disappeared after d 11 (data not shown). It must be noted that maternal RN produced in the uterus and decidua did not induce an activation of local RAS in the decidua of non-PAH mice (Fig. 3B
). All these results led us to hypothesize that the cell type-specific expression of RN and ANG in the labyrinth might contribute to the difference of their permeability with respect to their ability to enter the maternal circulation, and that causes contrasting phenotypes of the mother and fetus between the two lines of transgenic pregnant mice.
Differential Roles of Fetal-Derived hRN and hANG in the Labyrinth
To evaluate the different roles of fetal-derived hRN and hANG in the labyrinth, we compared plasma renin activity (PRA) in the cross-mating experiments (Fig. 7A
). In the nonpregnant state, no statistically significant difference was observed among the females, indicating the species specificity (mouse vs. human) of RN kinetics against ANG in transgenic mice. It is noteworthy that, compared with the nonpregnant state, PRA of both non-PAH and WT pregnant mice were induced about 5-fold higher at 15 d of gestation (P < 0.0001 by Students t test); however, no significant difference of PRA at d 15 was observed between the two lines. These data indicate that a general RAS activation was induced in non-PAH mice, as well as in WT pregnant mice (24). In both lines at 19 d of gestation the levels of PRA were decreased; on the contrary, a dramatic elevation of PRA was observed only in the PAH mice. Bohlender et al. (19) reported equivalent results regarding maternal blood pressure and maternal circulating levels of the RAS components in an analogous model for the rat with identical transgene overexpression. Combined with their results, the current data clearly demonstrated that hRN produced in trophoblast giant cells of the labyrinth is secreted into the maternal circulation and cleaves maternal hANG to generate excessive AI, whereas hANG produced in chorionic trophoblast cells and trophoblastic epithelium of the labyrinth could not enter the maternal blood system. Moreover, we confirmed the absence of hANG in the maternal circulation of non-PAH mice by the treatment of purified recombinant hRN (rhRN). As expected, the exogenous rhRN treatment did not enhance any AI production in the plasma of non-PAH mice, in contrast to a marked increase in AI levels observed in the plasma of nonpregnant hANG females (Fig. 7B
). All these findings strongly suggested that the distinct cell types that express RN and ANG in the labyrinth could cause the difference in their permeability with respect to the maternal circulation, and this impacts their differential roles in pregnancy.

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Fig. 7. Study of the Permeability of Feto-Placental hRN and hANG into the Maternal Circulation
A, Comparison of PRA among the pregnant mice. The lines of pregnant mice are indicated below. B, AI generation by the addition of excessive rhRN to the plasma of nonpregnant hANG females, non-PAH mice, and WT pregnant mice were measured by RIA. Note that this treatment did not induce a significant elevation in the non-PAH mice or in WT pregnant mice. Four to seven animals were used for each determination. Values are the means and SDs.
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DISCUSSION
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The circulatory anatomy of the mouse placenta is extremely similar to that of humans. In both species, maternal blood passes through spiral arteries into a space created by the conceptus that is densely filled with placental villi, termed a labyrinth in the mouse. Here, maternal blood irrigates the placental trophoblast directly (25). These trophoblast cells in humans and mice share common patterns of expression for genes that have been shown to be critical for either the development or function of the cells (26). The many obvious similarities of cellular and molecular features between the murine and human placenta make the mouse extremely useful as a model of human pregnancy (27).
Our previous studies in a transgenic mouse model used the mating of females expressing hANG with males expressing hRN and demonstrated that PAH and IUGR could be induced by the combined action of placental RN and maternal ANG (18, 20). During the cross-mating experiments, we observed that these phenotypic defects were not developed in the opposite type of mating combination, despite the overexpression of transgenes detected in the fetal placenta of both types of crossbreeding. In situ hybridization analysis provides important insights to an understanding of these discrepant phenotypes. The first and foremost, fetal-derived hRN and hANG genes exhibited distinct temporal and spatial patterns of expression in the surface of the labyrinth, suggesting their differential roles in the regulation of hemochorial blood flow through the placenta. Indeed, analysis using RIA clearly demonstrated that hRN, produced in trophoblast giant cells, was secreted into the maternal circulation and regulated maternal circulating and decidual RAS activation in the latter half of gestation, whereas hANG, produced in chorionic trophoblast cells and trophoblastic epithelium, could not pass through the selective barrier into the maternal blood system. We also found that hANG expression peaked in levels at midgestation and the same chorionic trophoblasts contained mRNA of MT1-MMP, which is essential for placental vascularization in humans (23). These results suggest that the feto-placental ANG production may be implicated in the formation of the labyrinthine structure, but further experiments are needed for decisive conclusion.
Next, we showed that hANG mRNA was expressed in the endoderm of visceral yolk sac and in the endothelial cells of fetal vessels. In addition, the overexpression of hRN and hANG was also detected in the fetal kidney and liver, respectively, of the non-PAH mice. Fetal expression of the RAS components during developmental stages is consistent with previous data of humans and rodents (28, 29, 30), indicating the potential contributions of fetal RAS to normal growth and development. It is noteworthy that, despite the up-regulation of fetal RAS, the mother and neonate did not display any obvious abnormalities when the hRN females were mated with the hANG males. These observations indicate that neither PAH nor IUGR could be induced by the fetal systemic RAS activation. At the same time, it is demonstrated that RN produced by placental trophoblasts interacts with maternal ANG to cause these defects in our transgenic mice model.
Histopathological examination revealed that placental abnormalities were observed in the PAH mice, but not in the non-PAH mice. In human placenta, the earliest stages of remodeling in uteroplacental arteries are independent of direct trophoblast invasion (31) and are considered to involve maternal activation of local RAS in the decidual arteries (15). Genetic studies showed that the maternal ANG T235 polymorphism associated with preeclampsia is tightly linked to a mutation in the ANG promoter A(-6) (32, 33), which may lead to elevated expression in human decidual spiral arteries (15). Because the reaction between RN and ANG is the initial and rate-limiting step of this enzymatic cascade, any abnormal elevation in local ANG expression would lead to abnormal elevation of local AII levels, and this may promote a relative increase of uteroplacental vascular resistance (34) and atherotic changes in spiral artery dilation (16). Recently, Adamson et al. (25) presented evidence that the dilation of the spiral arteries occurs in mice independently of invading trophoblast cells, similar to those described in humans. The current study showed that these vessels located within the decidua of PAH mice expressed hANG at high levels at 11 d of gestation. This result strongly confirms the involvement of maternal activation of local RAS in the decidual spiral arteries to the pathogenesis of complications of pregnancy.
Interestingly, our detailed analysis also revealed that adjacent localizations of hANG and MT1-MMP mRNAs could be detected at the junction between invasive trophoblast giant cells and maternal decidua, and in the maternal blood sinuses located close to the junctional zone. Several studies indicate that both matrix metalloproteinases and plasminogen activators have been implicated in a variety of developing processes in the mouse during implantation and placentation (35, 36, 37). A recent study also reported that AII activates gene expression of plasminogen activator inhibitor-1 through the AT1 receptor and inhibits human trophoblast invasion (38). It is thus possible that decidual ANG production may be involved in the regulation of trophoblast invasion and formation of maternal blood sinuses during decidualization. Further studies are required to define the type of decidual cells that express ANG. Such studies will bring further understanding to local function of the RAS in decidua.
We also found that maternally derived hANG was expressed in surrounding stromal cells and smooth muscle cells of large arteries located in the myometrium, suggesting its potential contributions to utero-placental blood flow. Collectively, all these findings lead us to speculate that locally generated ANG in the maternal decidua may interact with placental RN to activate the local RAS, and this could also trigger failed placental perfusion and function observed in the transgenic females with PAH and IUGR (15, 16).
Quite recently, we presented evidence that the genetic deletion of the maternal AII receptor type 1a (AT1a) had inhibitory effects on gestational hypertension and fetal growth restriction in transgenic females expressing hANG when mated with the hRN males (20). This supports the current data showing the importance of maternal RAS components to the pathogenesis of PAH and IUGR. In the transgenic mice model with homozygous disruption of maternal AT1a, an elevated level of AI was detected in the maternal circulation, as a result of combined action of placental RN and maternal ANG; despite this result, the mother did not show hypertension throughout pregnancy. It is interesting to note that their fetuses exhibited partially restricted growth in weight, compared with those delivered from WT pregnant mice, and from nontransgenic females with homozygous disruption of maternal AT1a when mated with the hRN males. These results suggest that the accelerated AII action through the feto-placental AT1a could directly evoke this fetal growth restriction, without AT1a actions in the maternal circulation. In human placenta, the elevated expression of AT1 protein was predominantly localized to the syncytiotrophoblasts in patients with preeclampsia (13), indicating the possible involvement of AII action through the feto-placental AT1 on the pathogenesis of complications of pregnancy.
The important concept to emerge consistently from our transgenic mouse model experiments is that the development of PAH and IUGR may require contributions of components of both the maternal and placental RAS genes. Data presented in this study clearly demonstrate that these defects can be mediated by fetal-derived placental hRN through its permeability with respect to the maternal circulation, not by feto-placental hANG production and not by fetal systemic RAS activation. Furthermore, the overproduction of hANG in maternal decidua could be important for the onset of placental hypoperfusion in abnormal pregnancy. These findings provide the first in vivo evidence that the cell type-specific expression of RN and ANG in the feto-maternal interface regulates their differential roles in pregnancy and also emphasize the role of tissue-based RAS in the pathogenesis of disease in human pregnancy.
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MATERIALS AND METHODS
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Animals
We previously created lines of transgenic mice carrying either the hRN gene (39) or the hANG gene (40) with their native promoters, respectively. These mice expressing each of the transgenes were normotensive, but the F1 progeny expressing both human genes exhibited a chronic hypertension (41). These transgenic mice and age-matched WT mice (C57BL/6J) at 2 to 4 months were used for all experiments. Care of experimental animals was within institutional guidelines approved by the Laboratory Animal Research Center at the University of Tsukuba.
Measurement of Blood Pressure
The systolic blood pressure of pregnant mice was measured by a programmable sphygmomanometer (BP-98; Softron, Tokyo, Japan) using the tail cuff method. Unanesthetized mice were introduced into a holder mounted in a thermostatically controlled warning plate and maintained at 37 C during measurement.
RNA Isolation and Northern Blot Analysis
Placental tissues were obtained from transgenic pregnant mice and WT control pregnant mice at 19 d of gestation, and fetal placenta was carefully separated from maternal decidua. In addition, fetal kidney and liver and maternal kidney were obtained from non-PAH mice, and maternal liver was obtained from PAH mice at 19 d of gestation. Total RNAs were isolated from these tissues using ISOGEN (NipponGene, Tokyo, Japan) based on the acid guanidium thiocyanate-phenol-chloroform extraction method. Total RNAs (20 µg) were denatured with glyoxal, separated by electrophoresis, and transferred to a nyrone membrane (Genesareen Plus: DuPont Merck Pharmaceutical Co., Boston, MA). Hybridization was carried out at 68 C or 65 C for 16 h in a 32P-labeled hRN cDNA probe (42) and a hANG cDNA probe (43), respectively. The membrane was washed as described previously (42) and subjected to autoradiography. Lanes of fetal placenta and decidua were exposed for 8 h (hRN) and for 4 h (hANG), and lanes of kidney and liver were exposed for 24 h (hRN) and for 40 min (hANG).
Preparation of Digoxigenin (DIG)-Labeled RNA Probe
DIG-labeled RNA sense and antisense probes were generated by in vitro transcription according to the manufacturers instructions (Roche Molecular Biochemicals, Indianapolis, IN) from subclones of the following cDNA: for hRN a 405-bp AatI-SacI fragment (44), and for hANG a 298-bp AatI-EcoRI fragment (43). The cDNA fragments of PL1, PL2, and MT1-MMP were obtained by RT-PCR using mRNA from murine placenta of 13 d of gestation for PL1 and PL2, and from immature rat ovary for MT1-MMP. Specific primers designed according to known nucleotide sequences were as follows. PL1, sense: 5'-TTGCTGCTGGTGTCAAGCCTAC-3'; and antisense: 5'-TCAATGTTAGCCTGAGACCTGTTG-3'; and PL2, sense: 5'-TCCAGAAAACAGCGAGCAAGTC-3'; and antisense: 5'-TGGGGGTAAGATGACAACTCAATC-3'; and MT1-MMP, sense: 5'-CGAGAACTTCGTGTTGCCTGATGAC-3'; and antisense: 5'-TATTCGCTGTCCACTGCCCTGAAC-3'.
In Situ Hybridization
Placental tissues of transgenic pregnant mice and WT control pregnant mice were obtained at the indicated times and were embedded in Tissue-Tec OCT compound (Miles Laboratories, Kankakee, IL) and frozen in a dry ice bath. Sections of 8 µm in thickness were cut on cryostat and mounted on silane-coated microscope slides. Hybridization was performed with DIG-labeled sense and antisense probes as described previously (45). Visualization of the signal was performed with nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate solution containing 0.24 mg/ml levamisole for 1 h (PL2) and overnight (the other signals). After color reaction, the sections were counterstained with methyl green. Sense probes gave negative results in all experiments, and both hRN and hANG signals were not detected in the placenta of WT pregnant mice.
Preparation of Plasma Fractions
Blood samples were withdrawn from transgenic and WT pregnant mice at the indicated times under pentobarbital anesthesia. Blood was collected into an ice-cold microcentrifuge tube containing disodium EDTA, which was then immediately centrifuged to isolate the plasma fraction. After addition of 5 mM phenylmethylsulfonylfluoride, 2 µg/ml antipain, and 4 µg/ml leupeptin to the plasma fraction, this fraction was stored at 80 C until needed.
Preparation of Tissue Extracts
Fetal placentas and deciduas were homogenized using Polytron PTA-7 in four volumes of PBS containing 5 mM EDTA, 5 mM phenylmethylsulfonylfluoride, 5 mM potassium tetrathionate, 2 µg/ml antipain, and 4 µg/ml leupeptin. After centrifugation at 100,000 x g for 1 h at 4 C, the supernatants were immediately used for measurement of RN, ANG, and AI contents or stored at 80 C until needed. The protein concentration of the tissue extract was measured by protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA).
Measurement of RN, Angiotensin Proteins, and PRA
Human active RN concentration was measured by means of commercially available RN immunometric assay kit (Diagnostics Pasteur, Marnes la Coquette, France), which is highly specific for activated hRN as this assay is based on the use of monoclonal antibodies (46, 47). Total RN concentration was defined as the RN concentration measured after prorenin activation. Activation of prorenin was performed with 100 µg/ml trypsin (Sigma Chemical Co., St. Louis, MO) at 24 C for 1 h, and then the reaction was stopped by incubation at 24 C for 15 min with 200 µg/ml soybean trypsin inhibitor (Sigma) (48). Inactive RN concentration was calculated as the difference between active and total RN concentration. Mouse RN did not cross-react with the human monoclonal antibodies used in this assay. The concentration of hANG was determined by measurement of the amount of AI released by the reaction with an excess of recombinant hRN as described previously (49). PRA was estimated by measuring the rate of AI formation (50). The concentrations of AI and AII were determined by RIA (51).
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ACKNOWLEDGMENTS
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We thank Dr. Yasuhiro Kon for providing helpful comments about placental morphology.
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FOOTNOTES
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This work was supported by The 21st Century Center of Excellence (COE) Program (to A.F.), Grant-in-Aid for Scientific Research (A) (to A.F.), and Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports, and Technology of Japan (to A.F.), "Research for the Future" Program [The Japan Society for the Promotion of Science (JSPS) (to A.F.), Grant RFTF 97L00804] (to A.F.), Research Grant 11C-1 for Cardiovascular Diseases (to A.F.), Comprehensive Research on Aging and Health from the Ministry of Health, Labour, and Welfare (to A.F.), and Fumi Yamamura Memorial Foundation for Female Natural Scientists (to E.T.-O.).
First Published Online February 3, 2005
Abbreviations: AI, Angiotensin I; AII, angiotensin II; ANG, angiotensinogen; DIG, digoxigenin; IUGR, intrauterine growth restriction; MT1-MMP, membrane type matrix metalloproteinase-1; PAH, pregnancy-associated hypertension; PL, placental lactogen; PRA, plasma renin activity; RAS, renin-angiotensin system; rhRN, recombinant hRN; RN, renin; WT, wild type.
Received for publication April 15, 2004.
Accepted for publication January 28, 2005.
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