K. Bridget Brosnihan

Angiotensin-converting enzyme 2 (ACE2) is a key enzyme of the renin-angiotensin system that influences the relative expression of angiotensin II (Ang II) and Ang-(1-7). Although ACE2 expression increases in normal pregnancy, the impact of ACE2 deficiency in pregnancy has not been elucidated. We determined the influence of ACE2 deficiency on circulating and tissue renin-angiotensin system components, fetal and maternal growth characteristics, and maternal hemodynamics (mean blood pressure and cardiac output) at day 18 of gestation. Gestational body weight gain was lower in the ACE2 knockout (KO) versus C57BL/6 (wild-type) mice (30.3±4.7 versus 38.2±1.0 g; P P P P P P P P <0.001). There was no change in the Ang-(1-7) levels in the KO placenta and virgin kidney. These results suggest that ACE2 deficiency and associated elevated placenta Ang II levels impact pregnancy by impairing gestational weight gain and restricting fetal growth.

Background: During preeclampsia, placental angiogenesis is impaired. Factors released from the placenta including vascular endothelial growth factor (VEGF), placental growth factor (PLGF), soluble VEGF receptor 1 (sFlt1), and soluble endoglin (sEng) are regulatory molecules of placental development and function. While the renin angiotensin system has been shown to regulate angiogenic factors in other research fields, these mechanisms have not been extensively studied during pregnancy. Methods: We evaluated the effects of angiotensin II (Ang II) and angiotensin-(1-7) [Ang-(1-7)] on the release of VEGF, PLGF, sFlt1, and sEng from placental chorionic villi (CV). CV were collected from nulliparous third-trimester normotensive and preeclamptic subjects. CV were incubated for 0, 2, 4, and 16 hours with or without Ang II (1 nM and 1 microM) or Ang-(1-7) (1 nM and 1 microM). The release of VEGF, PLGF, sFlt1, sEng, lactate dehydrogenase (LDH), and human placenta lactogen (HPL) was measured by ELISA. Results: The release of sFlt1, PLGF, sEng from normal and preeclamptic CV increased over time. Release of sFlt1 and sEng was significantly higher from preeclamptic CV. VEGF was below the detectable level of the assay in normal and preeclamptic CV. After 2 hours, sFlt1 release from normal CV was significantly inhibited with Ang II (1 nM and 1 microM) and Ang-(1-7) (1 nM and 1 microM). There was a time-dependent increase in HPL indicating that the CV were functioning normally. Conclusions: Our study demonstrates a critical inhibitory role of angiotensin peptides on sFlt1 in normal pregnancy. Loss of this regulation in preeclampsia may allow sFlt1 to increase resulting in anti-angiogenesis and end organ damage in the mother.

The pregnant female human angiotensinogen ( h AGN) transgenic rat mated with the male h renin ( h REN) transgenic rat is a model of preeclampsia with increased blood pressure, proteinuria, and placenta alterations of edema and necrosis. The reverse mating of female h REN×male h AGN does not show preeclamptic features. Because the placenta is well-recognized to be a key contributor to the preeclamptic syndrome, our hypothesis is that local angiotensin peptide concentrations found in the placenta and its associated mesometrial triangle of the preeclamptic transgenic rat differ from the reverse mating. We characterized the angiotensin peptide content and the mRNA expression of h REN and h AGN of the mesometrial triangle and the placenta. Three groups of pregnant rats from the matings (Sprague-Dawley×Sprague-Dawley, reverse mating, and female h AGN×male h REN) were studied on day 21 of gestation. In the h AGN× h REN transgenic rat, angiotensin II is significantly increased in the placenta and mesometrial triangle vs Sprague-Dawley (24.2±3.9 vs 8.6±1.5 pg/mg protein; 27.8±5.5 vs 5.6±1.3 pg/mg protein; P P <0.05) but unchanged in the mesometrial triangle (4.2±0.2 vs 8.6±1.5 pg/mg protein). The marked contrast in the expression of angiotensin II in the mesometrial triangle of the preeclamptic model vs the reverse mating suggests that local angiotensin II generated from the maternal parts of the uteroplacental unit may play a critical role in preeclampsia.

Previously, we demonstrated activation of the renin-angiotensin system in the fetal placental chorionic villi, but it is unknown whether the immediately adjacent area of the maternal uterine placental bed is regulated similarly. This study measured angiotensin peptides, renin-angiotensin system component mRNAs, and receptor binding in the fundus from nonpregnant subjects (n = 19) and in the uterine placental bed from normal (n = 20) and preeclamptic (n = 14) subjects. In the uterine placental bed from normal pregnant women, angiotensin II peptide levels and angiotensinogen, angiotensin-converting enzyme, angiotensin receptor type 1 (AT₁), AT₂, and Mas mRNA expression were lower as compared with the nonpregnant subjects. In preeclamptic uterine placental bed, angiotensin II peptide levels and renin and angiotensin-converting enzyme mRNA expression were significantly higher than normal pregnant subjects. The AT₂ receptor was the predominant receptor subtype in the nonpregnant fundus, whereas all angiotensin receptor binding was undetectable in normal and preeclamptic pregnant uterine placental bed compared with nonpregnant fundus. These findings suggest that the maternal uterine placental bed may play an endocrine role by producing angiotensin II, which acts in the adjacent placenta to vasoconstrict fetal chorionic villi vessels where we have shown previously that AT₁ receptors predominate. This would lead to decreased maternal-fetal oxygen exchange and fetal nutrition, a known characteristic of preeclampsia.

Pregnancy is characterized by an increase in many of the different components of the circulating renin—angiotensin system (RAS). However, the physiological mechanisms of stimulated RAS activity during pregnancy are unknown. Even less understood is how this system may be altered in pre-eclampsia, a hypertensive disorder of pregnancy. Additional studies have shown the presence of a local tissue specific RAS in the uteroplacental unit of normal and pre-eclamptic pregnancies. Differences in normal pregnant and pre-eclamptic RAS component regulation may provide insight into the mechanisms responsible for the clinical pathological features of pre-eclampsia. Specifically, this review summarizes the key findings in the circulating and uteroplacental RAS in normal and pre-eclamptic pregnancies.

Clinical trials revealed that estrogen may result in cardiovascular risk in patients with coronary heart disease, despite earlier studies demonstrating that estrogen provided cardiovascular protection. It is possible that the preexisting condition of hypertension and the ability of estrogen to activate the renin-angiotensin system could confound its beneficial effects. Our hypothesis is that the attenuation of estrogen to agonist-induced vasoconstrictor responses through the activation of nitric oxide (NO) synthase (NOS) is impaired by hypertension. We investigated the effects of 17β-estradiol (E₂) replacement in normotensive Sprague-Dawley (SD) and (mRen2)27 hypertensive transgenic (TG) rats on contractile responses to three vasoconstrictors, angiotensin II (ANG II), serotonin (5-HT), and phenylephrine (PE), and on the modulatory role of vascular NO to these responses. The aorta was isolated from ovariectomized SD and TG rats treated chronically with 5 mg E₂or placebo (P). The isometric tension of the aortic rings was measured in organ chambers, and endothelial NOS (eNOS) in the rat aorta was detected using Western blot analysis. E₂treatment increased eNOS expression in the SD and TG aorta and reduced ANG II- and 5-HT- but not PE-induced contractions in SD and TG rats. The inhibition of NOS with N^ω-nitro-l-arginine methyl ester enhanced ANG II-, 5-HT-, and PE-induced contractions in P-treated and ANG II and PE responses in E₂-treated SD and TG rats. Only the responses to 5-HT were augmented in hypertensive rats. In conclusion, this study shows that the preexisting condition of hypertension augmented the vascular responsiveness of 5-HT, whereas the attenuation of estrogen by ANG II and 5-HT vascular responses was not impaired by hypertension. The adrenergic agonist was unresponsive to estrogen treatment. The contribution of NO as a factor contributing to the relative refractoriness of the vascular responses is dependent on the nature of the vasoconstrictor and/or the presence of estrogen.

The chorionic villi in the placenta are responsible for the regulation of fetal oxygen and nutrient transport. Although the peripheral renin-angiotensin system is activated during normal pregnancy, the regulation of the local chorionic villi renin-angiotensin system remains unknown. Therefore, placental chorionic villous tissue was collected from nulliparous third-trimester normotensive or preeclamptic subjects and was analyzed for angiotensin peptide content, angiotensinogen, renin, angiotensin-converting enzyme (ACE), ACE2, neprilysin, angiotensin II type 1 (AT ₁ ), angiotensin II type 2, Mas receptor mRNAs, and angiotensin receptor density and subtype. Angiotensin II in chorionic villi was significantly higher in preeclamptic subjects, whereas angiotensin (1-7) was not different. Angiotensinogen and AT ₁ receptor gene expression was significantly higher in preeclamptic subjects. No differences were observed in renin, ACE, ACE2, or neprilysin gene expression. Mas receptor mRNA in preeclamptic subjects was decreased. The AT ₁ receptor was the predominant receptor subtype in normal and preeclamptic chorionic villi. There was no difference in the density of the AT _1, angiotensin II type 2, and angiotensin (1-7) receptors. These results indicate that enhanced chorionic villous expression of angiotensin II may result from increased angiotensinogen. Elevated angiotensin II, acting through the AT ₁ receptor, may favor vasoconstriction in placental chorionic villi and contribute to impaired fetal blood flow and decreased fetal nutrition observed during preeclampsia.

Angiotensin‐converting enzyme (ACE) and ACE2 and the AT₁ and AT₂ receptors are pivotal points of regulation in the renin–angiotensin system. ACE and ACE2 are key enzymes in the formation and degradation of angiotensin II (Ang II) and angiotensin‐(1–7)(Ang‐(1–7)). Ang II acts at either the AT₁ or the AT₂ receptor to mediate opposing actions of vasoconstriction or vasodilatation respectively. While it is known that oestrogen acts to downregulate ACE and the AT₁ receptor, its regulation of ACE2 and the AT₂ receptor and the involvement of a specific oestrogen receptor subtype are unknown. To investigate the role of oestrogen receptor‐α (ERα) in the regulation by oestrogen of ACE/ACE2 and AT₁/AT₂ mRNAs in lung and kidney, ovariectomized female mice lacking apolipoprotein E (ee) with the ERα (AAee) or without the ERα (ααee) were treated with 17β‐oestradiol (6 μg day⁻¹) or placebo for 3 months. ACE, ACE2, AT₁ receptor and AT₂ receptor mRNAs were measured using reverse transcriptase, real‐time polymerase chain reaction. In the kidney, 17β‐oestradiol showed 1.7‐fold downregulation of ACE mRNA in AAee mice, with 2.1‐fold upregulation of ACE mRNA in ααee mice. 17β‐Oestradiol showed 1.5‐ and 1.8‐fold downregulation of ACE2 and AT₁ receptor mRNA in AAee mice; this regulation was lost in ααee mice. 17β‐Oestradiol showed marked (81‐fold) upregulation of the AT₂ receptor mRNA in AAee mice. In the lung, 17β‐oestradiol treatment had no effect on AT₁ receptor mRNA in AAee mice, but resulted in a 1.5‐fold decreased regulation of AT₁ mRNA in ααee mice. There was no significant interaction of oestrogen with ERα in the lung for ACE, ACE2 and AT₂ receptor genes. These studies reveal tissue‐specific regulation by 17β‐oestradiol of ACE/ACE2 and AT₁/AT₂ receptor genes, with the ERα receptor being primarily responsible for the regulation of kidney ACE2, AT₁ receptor and AT₂ receptor genes.

Angiotensin-(1–7) is increased in the circulation during human pregnancy, but its functional role is unknown. Recent studies suggested that it opposes angiotensin II mediated vascular growth. Because angiogenesis is critical to normal embryonic development during human pregnancy, this study assessed the in vitro effects of angiotensin-(1–7) on human umbilical vein endothelial cell tube formation. The blocking effects of the angiotensin-(1–7) receptor antagonist, D-[Alanine⁷]-Ang-(1–7), and angiotensin II receptor AT₁ and AT₂ antagonists, losartan and PD123319, on tube formation were measured by counting tube branch points. Human umbilical vein endothelial cells were cultured in EGM-2 medium and treated with angiotensin-(1–7) (0.17 nM–17 μM) for 18 h. Angiotensin-(1–7) inhibited tube formation by 24% (P < 0.01) at all doses tested. Treatment with 1.7 μM angiotensin-(1–7) plus 17 μM D-[Alanine⁷]-Ang-(1–7) resulted in the reversal of angiotensin-(1–7) mediated inhibition of tube formation (P < 0.05). Losartan (17 μM) also reversed the angiotensin-(1–7) mediated inhibition of tube formation (P < 0.05). Tube formation was unaffected by PD123319. These results suggest that angiotensin-(1–7) has an anti-angiogenic effect on human umbilical vein endothelial cells through a unique AT_1–7 receptor that is sensitive to losartan, indicating that angiotensin-(1–7) may play an important role in the regulation of vascular growth in the placenta during pregnancy.