Effects of Circulating and Local Uteroplacental Angiotensin II in Rat Pregnancy
The renin-angiotensin (Ang) system is important during placental development. Dysregulation of the renin-Ang system is important in preeclampsia (PE). Female rats transgenic for the human angiotensinogen gene crossed with males transgenic for the human renin gene develop the PE syndrome, whereas those of the opposite cross do not. We used this model to study the role of Ang II in trophoblast invasion, which is shallow in human PE but deeper in this model. We investigated the following groups: PE rats, opposite-cross rats, Ang II–infused rats (1000 ng/kg per day), and control rats. Ang II infusion increased only circulating Ang II levels (267.82 pg/mL), opposite cross influenced only uteroplacental Ang II (13.52 fmol/mg of protein), and PE increased both circulating (251.09 pg/mL) and uteroplacental (19.24 fmol/mg of protein) Ang II. Blood pressure and albuminuria occurred in the models with high circulating Ang II but not in the other models. Trophoblast invasion increased in PE and opposite-cross rats but not in Ang II–infused rats. Correspondingly, uterine artery resistance index increased in Ang II–infused rats but decreased in PE rats. We then studied human trophoblasts and villous explants from first-trimester pregnancies with time-lapse microscopy. Local Ang II dose-dependently increased migration by 75%, invasion by 58%, and motility by 282%. The data suggest that local tissue Ang II stimulates trophoblast invasion in vivo in the rat and in vitro in human cells, a hitherto fore unrecognized function. Conceivably, upregulation of tissue Ang II in the maternal part of the placenta represents an important growth factor for trophoblast invasion and migration.
Various studies have implicated the renin-angiotensin (Ang) system in preeclampsia (PE).1–3⇓⇓ Female rats harboring the human angiotensinogen gene develop hypertension and proteinuria late in pregnancy when mated with male rats harboring the human renin gene; the result is a PE-like syndrome.4 However, instead of showing shallow trophoblast invasion associated with PE in humans, trophoblast invasion and spiral artery remodeling are actually increased in this animal model.4,5⇓ Trophoblast invasion is highly relevant in pregnancy. These fetal cells invade the maternal decidua and remodel the spiral arteries, a process that includes replacing the smooth muscle cell layer with fibrinoid material. The remodeling of the uterine spiral arteries causes the vessels to increase in diameter, allowing for an increase in blood flow to the intervillous space of the placenta.6 The role of Ang II in this process is unclear.
The hallmark of PE is a shallow trophoblast invasion accompanied by a reduced percentage of remodeled spiral arteries in the decidua and no remodeling in the inner myometrium.6 Preliminary observations in our transgenic rat model indicated that the opposite cross (OC), namely dams harboring a human renin gene mated with males harboring human angiotensinogen, did not develop hypertension or a PE-like syndrome.4 In addition to the circulating renin-Ang system, several authors have described a local, uteroplacental renin-Ang system in pregnancy.7–10⇓⇓⇓ However, the function of the uteroplacental renin-Ang system is unknown. Consistent with the observations in rat pregnancy, we showed earlier that components of the tissue renin-Ang system are substantially higher in the maternal part of the human placenta (the decidua), than in the fetal part (placenta), suggesting a role for local Ang II in the trophoblast-decidua interaction.11
We investigated the effects of uteroplacental Ang II and circulating Ang II in rat pregnancy in more detail. We studied our conventional PE-syndrome cross, the OC, and chronic Ang II–infused pregnant rats and appropriate control rats. We also extended our observations to in vitro Ang II–related effects on cells derived from human trophoblasts and villous explants from first-trimester pregnancies. Our data show that uteroplacental Ang II has opposite in vivo effects on trophoblast invasion, migration, uteroplacental vascular remodeling, and maternal endothelial dysfunction compared with circulating Ang II. Local Ang II could serve as a potent regulator promoting trophoblast migration and invasion early in pregnancy.
Details on the TGR models (PE and OC) have been published previously.4,12⇓ On day 9 of gestation, Sprague-Dawley (SD) dams were randomly assigned to 1 of 2 experimental groups: SD plus vehicle, which served as the control group (n=9) or SD plus Ang II (n=8), at a dose of 1000 ng/kg per min (Ang II). The animals were followed to term (day 21), anesthetized, and then euthanized. Doppler studies were performed as described earlier.13 Peak systolic velocity (PSV) and end-diastolic velocity (EDV) were measured from 3 cardiac cycles. The resistance index (RI=[PSV−EDV]/PSV) was calculated when EDV was >0 to quantify the pulsatile arterial blood velocity waveforms.
Tissue sections were immediately snap frozen in liquid nitrogen and homogenized as described previously.4,11,13⇓⇓ Tissue Ang II and circulating Ang II were measured by radioimmunoassay.7 Tissue preparations and immunostaining protocols were performed as described earlier.5,14⇓ For the quantitative analysis of depth of invasion and spiral artery remodeling, the KS-400 image analysis system (Carl Zeiss) was used, and the length of each traced line was measured and calculated in relation to spiral artery lumen contour. More detailed information is given in the online Data Supplement (please see http://hyper.ahajournals.org).
SGHPL-4 cells (derived from primary human first-trimester extravillous trophoblasts transfected with the early region of SV40, known previously as MC418) were cultured as described earlier.15 The effect of 48-hour Ang II stimulation in matrigel on the invasive trophoblast outgrowth was investigated as described earlier.16 Human placental villous tissue explants from first-trimester pregnancies were established and characterized as described previously.17 Migration experiments were conducted with collagen-coated chambers after 72-hour Ang II exposure. More detailed information is given in the online Data Supplement.
Data are presented as mean±SEM. We tested Kolmogorov-Smirnov for distribution. For group differences we tested 1-way ANOVA with Bonferroni post hoc test, Dunnett T3, or Kruskal-Wallis test and Mann-Whitney U test, as appropriate. A value of P<0.05 was considered statistically significant.
Circulating and Local Uteroplacental Ang II
Circulating Ang II was increased in PE rats and in rats after Ang II infusion (Figure 1A). OC rats had similar plasma Ang II levels as SD. In contrast to the circulating plasma, local uteroplacental tissue concentration of Ang II was increased in both PE and OC rats (Figure 1B). Ang II infusion did not lead to an increase in uteroplacental Ang II. Thus, PE is a rat model with high uteroplacental and circulating Ang II levels. OC results in solely high uteroplacental Ang II values at term and normal circulating values. Human renin and angiotensinogen (hAogen) mRNA expressions were significantly elevated in the OC and PE uteroplacental units and were undetectable in SD and Ang II (Figure S1, available in the online Data Supplement). hAogen expression was significantly higher in PE compared with OC, especially in the mesometrial triangle. Ang II infusion raised the circulating Ang II values but not the uteroplacental Ang II concentrations.
Blood Pressure, Albuminuria, and Intrauterine Growth Retardation
Telemetric blood pressure measurements showed that mean arterial blood pressure significantly increased PE and Ang II and decreased in OC in the last third of pregnancy. Blood pressure was significantly increased in PE and Ang II compared with SD. Mean arterial blood pressure was significantly decreased in OC versus control (Figure 2A). Albuminuria was present in PE and Ang II–infused rats, but not in OC or SD rats, in the last third of pregnancy (Figure 2B). Placental weight was lower in PE and Ang II–infused rats. OC rats had placental weights no different from SD rats (Figure 2C, left). Fetal body weight was reduced in PE, OC, and Ang II–infused rats (Figure 2C, middle). To further characterize intrauterine growth retardation (IUGR), we investigated the brain:liver ratio in the fetuses. In PE and Ang II–infused rats, we found a reduced liver and preserved brain weight, leading to an increased brain:liver ratio consistent with IUGR (Figure 2C, right). The OC rats had a normal brain:liver weight ratio and a normal placenta weight, so that the criteria for IUGR were not fulfilled, although these rats had pups small for gestational age compared with SD rats. These data indicate that PE-like features and IUGR were only present in the groups with high circulating Ang II levels. However, slow fetal growth without signs of IUGR took place when uteroplacental Ang II was elevated, even with normal circulating Ang II levels.
Trophoblast Invasion and Spiral Artery Vascular Remodeling
Endovascular trophoblast invasion was evaluated in the mesometrial triangle in 3 zones according to the immunohistograph picture, which was stained with cytokeratin 7 to identify trophoblasts (Figure 3A, left) and as described earlier.5,13,18⇓⇓ We showed earlier that deep endovascular trophoblast invasion was significantly increased in PE and OC rats, resulting in altered vascular remodeling of spiral arteries (Figure 3A, right).5,13⇓ In contrast, Ang II infusion resulted in a reduced deep-trophoblast invasion in the mesometrial triangle compared with SD controls (Figure 3B, left). Examples of reduced trophoblast invasion by Ang II infusion compared with SD are shown (Figure 3B, right). In accord with alterations in trophoblast invasion, persistence of vascular smooth muscle cells (VSMC) was increased in Ang II–infused compared with SD controls (Figure 3C). We measured the functional consequences of the altered trophoblast and VSMC expression by B-mode Doppler ultrasound (Figure 3D). The measurements showed increased resistance index with Ang II infusion compared with SD controls, indicating that placental blood flow had decreased. We found a decrease in the resistance index in PE rats and OC rats, consistent with our earlier report.13
Invasion, Migration, and Motility of Trophoblasts
We next investigated human trophoblasts from fist-trimester pregnancies to determine the effect of uteroplacental Ang II. We stimulated SGHPL-4 cells, which were derived from primary human first-trimester extravillous trophoblasts. The cells were treated with 2 concentrations of Ang II and were studied with time-lapse microscopy (Figure 4A). Ang II dose-dependently increased the various aspects of invasion (showing more and deeper invasion) and motility. The effects of Ang II were blocked completely with losartan. To determine whether Ang II has an effect on the migration, villous explants from first-trimester normal pregnancies were used for in vitro invasion assay experiments in collagen-coated chambers (Figure 4B). Ang II for 72 hours resulted in a strong increase in the migration of extravillous trophoblasts cells compared with control cells. These results were reproducible in 3 separate experiments.
The novel finding in our study was that circulating Ang II reduced trophoblast invasion and vascular remodeling in the uteroplacental unit. We found opposite effects of increased circulating and uteroplacental Ang II in pregnant rats. Ang II–infused rats exhibited an increased blood pressure in the third trimester, albuminuria, and IUGR. The OC model, in which only uteroplacental tissue Ang II is increased, showed increased trophoblast remodeling. Blood pressure was lower than in controls, no albuminuria was present, and pup weights were reduced. In the PE cross, in which circulating and tissue Ang II are increased, we observed hypertension in the last trimester, albuminuria, IUGR, increased trophoblast invasion, and uteroplacental vascular remodeling. Finally, by applying Ang II directly to human first-trimester trophoblasts and villous explants, we found that invasion, migration, and motility were increased in response to local Ang II. We believe that our results show a different and unique function for uteroplacental Ang II in pregnancy. Uteroplacental Ang II but not circulating Ang II increased trophoblast invasion and vascular remodeling.
Several studies have shown the presence of a local tissue-specific renin-Ang system in the uteroplacental unit of normal pregnancies.2,7–10⇓⇓⇓⇓ Estrogen increases both tissue and circulating levels of angiotensinogen and renin.7 Plasma Ang II, together with angiotensinogen and plasma renin activity, is increased during normal pregnancy.19 However, the physiological function of a stimulated uteroplacental renin-Ang system during pregnancy is unknown. Our results of an increased trophoblast invasion by tissue Ang II are in line with the human results that we reported earlier in that increased renin, angiotensinogen, Ang-converting enzyme (ACE), and Ang II are substantially increased in the maternal part of the uteroplacental unit, the deciduas, compared with the fetal part of the placenta.11 We now suggest that the maternal-fetal Ang II gradient contributes to trophoblast invasion and migration. Rosenfeld and Gant20 reported that Ang II infusion increased blood pressure in sheep but reduced uteroplacental perfusion, which was measured invasively with chronically implanted flow probes around both major uterine arteries. Their experiments suggested an autoregulation of uterine blood flow. Earlier, Ferris et al21 showed that an increase of uterine renin production resulted in an increase in uterine perfusion in rabbits. Uterine perfusion was determined with radioactive microspheres lodged in the uterus and placenta after injection in the left ventricle in that study. Li et al22 were the first to show that endocrine-active decidual cells express both renin and angiotensinogen in the human uteroplacental unit. They suggested that decidual cells regulate a tissue renin-Ang system expression in the endometrium. Anton and Brosnihan7 were the first to suggest a different regulation for circulating and uteroplacental Ang II in PE. They observed contrasting changes for Ang II levels in the circulation and chorionic villi in human preeclamptic pregnancy. Although circulating Ang II was significantly decreased, the expression of local tissue Ang II was augmented in the placenta of the preeclamptic women. Uteroplacental Ang II in the OC and PE groups was elevated because the expression of both human renin gene and hAogen mRNA in the uteroplacental unit was increased. hAogen was significantly higher in PE, indicating that hAogen concentrations were responsible for the different Ang II levels in OC and PE rats.23 Morgan et al24 were able to show increased expression of an angiotensinogen variant in human decidual spiral arteries that are involved in the generation of atherotic (a histopathologic term applied to PE-related vascular changes resembling atherosclerosis) lesions overrepresented in PE.
Trophoblast cells of the human placenta proliferate, migrate, and invade the pregnant uterus and remodel its vasculature. Trophoblast-associated spiral artery remodeling is an important uterine adaptation mechanism of human pregnancy, allowing an adequate maternal blood supply to the placenta. Few factors have been shown to promote trophoblast invasion and survival of these fetus-derived cells in the maternal surroundings, including corticotropin-releasing hormone, insulin-like growth factor binding protein 1, and various cytokines, such as interleukin 1.25 The role of many growth factors and proto-oncogenes has not yet been thoroughly studied in the decidua.
We found that fetal weight was decreased in the Ang II–infused group compared with controls. Similar to patients with PE, we observed a shallow trophoblast invasion, as well as persistence of VSMCs and reduced spiral artery remodeling in the group with chronic Ang II infusion. The increased resistance indicates a disturbed placental function with reduced transformation of the arterioles into a highly dilated vessel. In accordance with the human pathology in PE, the increased vascular resistance led to IUGR and decreased birth weight in this group. The pathophysiological processes underlying IUGR are highly complex and incompletely understood. The finding that the fetal weight was decreased in the OC group appears surprising, because trophoblast invasion is increased and vascular resistance decreased. The pups exhibited lower birth weight, but not IUGR, suggesting a different mechanism compared with the Ang II–infused group that did exhibit IUGR. In earlier studies, we showed that atherosis is present in OC, which also might contribute to impaired fetal growth.4,5⇓ We noted focal necrosis in the arterial wall in 25% of spiral arteries in that study. The atherosis in these models is similar to the acute atherosis associated with placental infarcts observed in uteroplacental spiral arteries of preeclamptic women.26 The impact of this vasculopathy on fetal growth is unclear.6,27⇓ Shibata et al28 showed that Ang II suppresses Na+-K+-ATPase in human placental villi, consistent with possible adverse effects of enhanced placental Ang II on fetal growth. These findings are consistent with those of our OC.
We showed that local Ang II caused human trophoblasts to migrate and invade, similar to the trophoblast behavior in the PE and OC models, where uteroplacental Ang II is elevated. These data could suggest similar effects and functional roles of local uteroplacental Ang II in our rat model and in the decidua of pregnant women. Furthermore, our findings could suggest that Ang II plays a central role in signaling trophoblast invasion and, ultimately, spiral artery remodeling. Ang II induces migration of various cells, including dendritic cells, VSMCs, and glomerular mesangial cells.29 The results in trophoblasts are controversial. Xia et al30 found that Ang II inhibits migration of the trophoblast cell line, HTR-8, via plasminogen activator inhibitor 1 activation. Ishimatsu et al31 found the opposite. They showed that direct-application Ang II induces migration in human cytotrophoblast-like choriocarcinoma cell lines. Local Ang II also influences endothelial cadherin, platelet-endothelial adhesion molecule 1, vascular endothelial adhesion molecule 1, and α4 integrins. These factors are important in facilitating the adhesion-phenotype switch in trophoblasts, which is required for successful endovascular invasion and normal placentation.32 Taken together, these data suggest that local Ang II is an important uteroplacental factor regulating the temporal and spatial regulation of trophoblast invasion in a paracrine way.
Circulating Ang II levels in the PE cross increased between day 9 and day 15, exactly when we started the Ang II infusion in the Ang II–infused group. However, a shorter time of exposure to Ang II in the Ang II–infused group compared with the transgenic group cannot be excluded as a variable contributing to the observed phenotypes. We also excluded the possibility that proteases are dysregulated in the Ang II–infused group. Aminopeptidase A, Prolyl-carboxypeptidase, Neprilysin, ACE-1, and ACE-2 were not induced in the Ang II–infused group compared with controls (data not shown). Aminopeptidase A, ACE-1, and ACE-2 were upregulated in OC and PE compared with control and Ang II (data not shown).
Ours and other laboratories have described activating autoantibodies directed at the Ang II type 1 receptor in women with human PE.3,33⇓ We made similar observations in PE rats.4,34⇓ The role of these antibodies is still unclear. However, upregulation of the decidual Ang II type 1 receptor is also present in PE.11,35⇓ Both events could represent a possible physiological adaptation, suggesting the necessity of increased Ang II–like activity in promoting trophoblast invasion for spiral artery remodeling. The antibodies could represent malfunction of this mechanism resulting in PE in some women.
The rodent transgenic renin/angiotensinogen model was originally described in mice by Takimoto et al36 Hypertension, proteinuria, and IUGR described in that study were very similar to the rat PE model that we report. However, placental abnormalities were different in the 2 species. Takimoto et al36 detected necrosis in spongiotrophoblasts and decidual cells, edematous enlargement, and congestion in chorion cells. In a follow-up study, the group showed that the interaction between fetal vasculature and maternal blood canal in the labyrinth was distorted and the expression patterns of key molecules in neovascularization were dysregulated.37 The maternal plasma level of sFlt-1 was significantly increased after midterm in that model.37 In contrast to the mouse, the rat develops deep endovascular and interstitial trophoblast invasion beyond the decidua into the mesometrial triangle, showing a deep endovascular trophoblast-associated spiral artery remodeling. Thus, we can investigate the labyrinth placenta and the mesometrial triangle separately. sFlt-1 is not dysregulated in the rat placenta or in the maternal serum in PE rats.13 In spite of the species differences, both models underscore the notion that cell-specific expression of renin and angiotensinogen in the fetomaternal interface strongly influences IUGR and placenta development.
A limitation in our study is that an ACE inhibitor and/or Ang II type 1 receptor blocker was not tested in our models. Saito et al showed that genetic deletion of the maternal Ang II type 1a receptor, and cross-breeding with the transgenic mouse model significantly ameliorated the phenotype in their mice.38 The role of Ang II inhibition on trophoblast function, vascular remodeling in the uteroplacental unit, and IUGR requires substantial further study in animal models, particularly because human studies along these lines cannot be performed.
Uteroplacental Ang II promoted trophoblast migration and invasion in rats and humans. This effect could mediate a cross-talk between invasive trophoblasts and the maternal endometrium. Such a paracrine Ang II function in normal pregnancy could also be further activated in pregnancies with abnormal placentation that exhibit shallow trophoblast invasion, as in PE. The concept of a fetal-maternal Ang II gradient warrants further investigations, such as the determination of Ang II in the amniotic fluid.
We thank Jutta Meisel, Juliane Anders, Raika Langanki, May-Britt Koehler, and Gabriele N′diaye for their excellent technical assistance.
Sources of Funding
The Deutsche Forschungsgemeinschaft supported R.D. (631/7-1). D.N.M. is a Helmholtz fellow.
L.H. and F.H. contributed equally to this work.
- Received January 26, 2010.
- Revision received February 16, 2010.
- Accepted May 12, 2010.
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