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An essential role for angiotensin II type 1a receptor in pregnancy-associated hypertension with intrauterine growth retardation

TOMOKO SAITO^*,, JUNJI ISHIDA^*,, ERIKO TAKIMOTO-OHNISHI^*,, SHOKO TAKAMINE^*,, TAKU SHIMIZU^*,, TAKESHI SUGAYA^*,, HIDEKI KATO^*,,||, TOSHIKI MATSUOKA^*,, MASAOMI NANGAKU^||, YASUHIRO KON^¶, FUMIHIRO SUGIYAMA^,, KEN-ICHI YAGAMI^, and AKIYOSHI FUKAMIZU^*,,,2

^* Institute of Applied Biochemistry,
Aspect of Functional Genomic Biology, Tsukuba Advanced Research Alliance (TARA),
Institute of Basic Medical Sciences,
Laboratory Animal Resource Center, University of Tsukuba, Tsukuba, Ibaraki, Japan;
^|| Division of Nephrology and Endocrinology, University of Tokyo School of Medicine, Bunkyo, Tokyo, Japan; and
^¶ Laboratory of Anatomy, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido, Japan

²Correspondence: 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

SPECIFIC AIMS

We previously reported that mice, made by cross-mating normotensive human angiotensinogen transgenic (hAG^+/+) female mice with normotensive human renin transgenic (hRN^+/+) male mice, show hypertension in late pregnancy, due to excessive angiotensin I (AI), the precursor of angiotensin II (AII), generated by secreting human renin (hRN) from the fetal side to the maternal circulation. In the present study, we created a new strain of mice, in which the endogenous mouse AT1a gene (mAT1a) was genetically deleted from hAG^+/+ mice, hAG^+/+/mAT1a^-/- mice, and analyzed the contribution of angiotensin type-1 receptor (AT1a) to pregnancy-associated diseases such as hypertension, cardiac hypertrophy, placental defects, and intrauterine growth retardation (IUGR) found in pregnant hypertensive hAG^+/+ mice.

PRINCIPAL FINDINGS

1. Hypertension in pregnancy was mediated by AT1a
When pregnant hAG^+/+ and hAG^+/+/mAT1a^-/- mice that possess the hAG gene, mated with hRN^+/+ male mice, the elevated levels of AI and hRN were detected in the maternal blood on gestation day 19, suggesting that hAG in the maternal sides reacted to hRN derived from the fetal side. Under the conditions in which the renin-angiotensin system (RAS) was activated in the maternal circulation of both pregnant hAG^+/+ and hAG^+/+/mAT1a^-/- mice, to ascertain whether the AT1a mediated-pathways participate in elevating the blood pressure of our pregnant hAG^+/+ mice, we conducted a time-course measurement of systolic blood pressure during and after pregnancy for the four groups; pregnant WT, mAT1a^-/-, hAG^+/+, and hAG^+/+/mAT1a^-/- mice (Fig. 1 A).

View larger version (69K): [in this window] [in a new window]   Figure 1. Pathological changes of pregnant hAG+/+ mice and normal phenotypes of pregnant hAG+/+/mAT1a-/- mice in maternal body. All female mice mated with hRN+/+ male mice except for WT female mice, which mated with WT male mice. A) Systolic blood pressure values (mmHg) of pregnant hAG+/+/mAT1a-/- (n =10), pregnant hAG+/+ (n =7), mAT1a-/- (n =8), and WT (n =9) mice during and after pregnancy were represented. Day 1 corresponds to vaginal plug detection and delivery is on days 20 or 21. Filled circles, pregnant hAG+/+/mAT1a-/- mice; squares, pregnant hAG+/+ mice; empty circles, mAT1a-/- mice; triangles, WT mice. B) Heart weights of female mice in pregnancy on gestation day 19. Each heart weight was measured as wet weight. Horizontal bars represent mean values. *P < 0.001 compared with WT mice. C–E) Histopathological analysis of female mice heart on gestation day 19. Hematoxylin and eosin stain in (C) and (D), and Masson’s trichrome stain in (E). Scale bars, 1.0 mm in (C), and 5 µm in (D) and 10 µm in (E). Arrows indicate necrosis and vacuoles in the nucleus in (D). F and G) Histopathological analysis of female mice placenta on gestation day 19. Each placenta was stained with hematoxylin and eosin. Scale bars, 0.8 mm in (F) and 10 µm in (G). Arrows indicate the edematous enlargement in (F) and (G).

As shown previously, when hAG^+/+ female mice mated with hRN^+/+ male mice, the blood pressure of pregnant hAG^+/+ mice began to increase on gestation day 14, and continued to rise until the day before delivery, and returned to the normal level, as seen on gestation day 1 after delivery (Fig. 1A ; squares). By deleting maternal AT1a from pregnant hypertensive hAG^+/+ mice, pregnant hAG^+/+/mAT1a^-/- mice (filled circles) did not develop hypertension in late pregnancy like pregnant WT (triangles) and mAT1a^-/- mice (empty circles).

2. AT1a contributed to cardiac hypertrophy in pregnant hAG^+/+ mice
We next analyzed the effect of AT1a deficiency on concentric cardiac hypertrophy found in pregnant hAG^+/+ mice. While there was a significant increase in heart/body weight (BW) ratios of pregnant hAG^+/+ mice mated with hRN^+/+ male mice in comparison with that of pregnant WT and mAT1a^-/- mice on gestation day 19 (57.1±6.74 mg/10 g BW vs. 31.3±0.770 mg/10 g BW and vs. 31.0±3.71 mg/10 g BW, respectively), the heart/body weight ratios of pregnant hAG^+/+/mAT1a^-/- mice (35.4±8.17 mg/10 g BW) were similar to those of pregnant WT and mAT1a^-/- mice (Fig. 1B ). In pregnant hAG^+/+ mice, the necrosis including the obscure structure of nucleus and vacuoles in the myocyte (Fig. 1D , arrows), and the myocardial fibrosis around the vascular wall in preg-nant hAG^+/+ mice (Fig. 1E ), but not seen on gestation day 19 in pregnant hAG^+/+/mAT1a^-/- mice as well as in pregnant WT and mAT1a^-/- mice (Fig. 1C-E ).

3. IUGR and placental defects were induced by AT1a pathways
In all pregnant hAG^+/+ mice mated with hRN^+/+ male mice examined, the placental morphological changes including necrosis and edematous enlargement in the layer of labyrinth and IUGR were also found (Fig. 1F, G , arrows, and Fig. 2 A, in pregnant hAG^+/+ mice). In the placenta, these pathological phenotypes observed in pregnant hAG^+/+ mice were not seen in pregnant hAG^+/+/mAT1a^-/- mice as well as in pregnant WT and mAT1a^-/- mice (Fig. 1F, G ). The weights of fetuses from pregnant hAG^+/+ mice were significantly less than those from pregnant WT and mAT1a^-/- mice (hAG^+/+, 0.773±0.096 g; WT, 1.21±0.081 g; mAT1a^-/-, 1.23±0.079 g; P<0.001) (Fig. 2B ). In contrast, the fetal weights of pregnant hAG^+/+/mAT1a^-/- mice were sufficiently improved in comparison with those of pregnant hAG^+/+ mice (hAG^+/+/mAT1a^-/-, 1.06±0.096 g; P<0.001), but still significantly smaller than those from pregnant WT and mAT1a^-/- mice (P<0.001).

View larger version (61K): [in this window] [in a new window]   Figure 2. Pathophysiological analysis in fetus. A) Comparison of growth among neonates of each group (0 days). Scale bars, 5mm. B) Comparison of body weights among fetuses of each group. Fetuses were obtained from mice killed on gestation day 19. Body weights were compared among eight litters per dam of each group. Data are means ± SD; n = 16-56 fetuses per group. *P < 0.001 vs. WT mice.

4. IUGR was prevented by limited administration of AT1 antagonist during late pregnancy
Because the activation of AT1a-mediated pathways in late pregnancy of hAG^+/+ female mice mated with hRN^+/+ male mice induced hypertension, organ damages, and IUGR, it is likely that the repression of AT1a pathways ameliorates these phenotypes. Therefore, to analyze the effect of the short term suppression of AT1 signaling, we administered TCV-116 (5 mg/kg/day), a specific AT1 antagonist, to hAG^+/+ female mice mated with hRN^+/+ male mice on gestation day 18 and 19. It was effective for pregnant hAG^+/+ mice to lower the elevated blood pressure and to improve the delayed delivery. Furthermore, the birth weight of neonates from pregnant hAG^+/+ mice recovered to the level of neonates from pregnant WT mice by the short term administration of TCV-116.

CONCLUSIONS

In rodents, AT1 exists as two isoforms, namely AT1a and AT1b. AT1a, but not AT1b, mainly plays an essential role in the elevation of blood pressure. In this study, although both pregnant hAG^+/+ and hAG^+/+/mAT1a^-/- mice mated with hRN^+/+ male mice generated a considerable amount of angiotensins in the maternal circulation, the latter did not exhibit hypertension in late pregnancy. Thus, it is clear that the elevation of blood pressure in pregnant hAG^+/+ mice was induced by the activation of AT1a in late pregnancy.

In pregnant hAG^+/+ mice mated with hRN^+/+ male mice, cardiac hypertrophy with fibrosis and necrosis, and edematous enlargement and necrosis in the placenta were observed, suggesting the dysfunction of heart and placenta. These changes with hypertension were probably induced by the excess of AII which was generated in the maternal circulation from gestation day 13. As these morphological changes were improved in pregnant hAG^+/+/mAT1a^-/- mice, AT1-mediated pathways including hypertension contributed to the cardiac remodeling and placental defects in pregnant hAG^+/+ mice.

In this study, we revealed that all fetuses from pregnant hAG^+/+ mice mated with hRN^+/+ male mice induced IUGR accompanied with placental morphological changes. One of the causes of IUGR in preeclampsia has been considered to be the poor placental perfusion by both aplasia in the placenta and hypertension, suggesting a deleterious role for AT1 signaling against fetal growth. In this point, the dramatic improvement of IUGR by the genetic deletion of AT1a demonstrated the association of maternal AT1a with the development of IUGR.

Owing to the long term and high dose administration of ACE inhibitors and AT1 antagonists during pregnancy, fetal toxic effects such as limb malformation, pulmonary hypoplasia, and hydronephrosis have been observed. In our present study, by administration of AT1 antagonist in the limited term and the selective inhibition of AII actions, blood pressure in pregnant hAG^+/+ mice decreased, and fetal growth in pregnant hAG^+/+ mice was recovered without the toxic effects. Therefore, an adequate repression of AT1-mediated pathways in late pregnancy might be effective for improving the pathophysiological phenotypes in pregnant hAG^+/+ mice.

In conclusion, by the combined usage of transgenic and knockout mice, we provided here the first evidence that the pathophysiological importance of AT1a-mediated pathways for pregnancy-associated hypertension (Fig. 3 ) and suggested a therapeutic aspect under the appropriate timing and limited period blockage of AT1 signaling at late pregnancy for both mothers and fetuses in preeclampsia.

View larger version (34K): [in this window] [in a new window]   Figure 3. Schematic diagram of pathophysiology in pregnancy-associated hypertensive mice. When hAG+/+ female mice mated with hRN+/+ male mice, hypertension, cardiac hypertrophy, placental defects and IUGR were observed in pregnant hAG+/+ mice. It is suggested that these phenotypes in pregnancy-associated hypertensive mice were induced due to the activation of AT1a pathways by an excess of AII, which is generated from maternal angiotensinogen derived from fetal hRN.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/1096/fj.03-0321fje;

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