Uterine artery dysfunction in pregnant ACE2 knockout mice is associated with placental hypoxia and reduced umbilical blood flow velocity

Abstract

Angiotensin-converting enzyme 2 (ACE2) knockout is associated with reduced fetal weight at late gestation; however, whether uteroplacental vascular and/or hemodynamic disturbances underlie this growth-restricted phenotype is unknown. Uterine artery reactivity and flow velocities, umbilical flow velocities, trophoblast invasion, and placental hypoxia were determined in ACE2 knockout (KO) and C57Bl/6 wild-type (WT) mice at day 14 of gestation. Although systolic blood pressure was higher in pregnant ACE2 KO vs. WT mice (102.3 ± 5.1 vs. 85.1 ± 1.9 mmHg, n = 5-6), the magnitude of difference was similar to that observed in nonpregnant ACE2 KO vs. WT mice. Maternal urinary protein excretion, serum creatinine, and kidney or heart weights were not different in ACE2 KO vs. WT. Fetal weight and pup-to-placental weight ratio were lower in ACE2 KO vs. WT mice. A higher sensitivity to Ang II [pD2 8.64 ± 0.04 vs. 8.5 ± 0.03 (-log EC50)] and greater maximal contraction to phenylephrine (169.0 ± 9.0 vs. 139.0 ± 7.0% KMAX), were associated with lower immunostaining for Ang II receptor 2 and fibrinoid content of the uterine artery in ACE2 KO mice. Uterine artery flow velocities and trophoblast invasion were similar between study groups. In contrast, umbilical artery peak systolic velocities (60.2 ± 4.5 vs. 75.1 ± 4.5 mm/s) and the resistance index measured using VEVO 2100 ultrasound were lower in the ACE2 KO vs. WT mice. Immunostaining for pimonidazole, a marker of hypoxia, and hypoxia-inducible factor-2α were higher in the trophospongium and placental labyrinth of the ACE2 KO vs. WT. In summary, placental hypoxia and uterine artery dysfunction develop before major growth of the fetus occurs and may explain the fetal growth restricted phenotype.

Keywords: angiotensin II; fetal growth restriction; hypoxia; vascular reactivity; vasoconstriction.

Fig. 1.

Uterine artery responses in angiotensin-converting enzyme 2 knockout (ACE2 KO) and C57Bl/6 mice at day 14 of gestation. Stress-stretch response (A) and its rate constant (B) (n = 4 in each group); vasodilatory response to acetylcholine (C) and endothelial NOS immunostaining (D) (n = 4 in each group); contraction to phenylephrine (E) (n = 5–7). Data are means ± SE. *P < 0.05 vs. C57Bl/6.

Fig. 2.

Uterine arteries responses to Ang II (A and B) and semiquantitative analysis of angiotensin receptor (ATR) expression (C) in uterine arteries of ACE2 KO and C57Bl/6 mice at day 14 of gestation. A: concentration-dependent response to Ang II. B: maximal response to Ang II (10⁻⁸ M): Ang II alone, after preincubation with Ang-(1–7) (10⁻⁶M) or a combination of Ang-(1–7) with AT_1–7/MAS receptor antagonist [d-Ala⁷]-Ang-(1–7) (10⁻⁵ M) for 20 min. Data are means ± SE. *P < 0.05 vs. C57Bl/6; #P < 0.05 vs. ACE2 KO control (Ang II contraction); n = 5–7 for vascular responses; n = 4 in each group for immunostaining analysis.

Fig. 3.

Images (A) and semiquantitative analysis (B) of morphological markers of arterial stiffness (PSR), elasticity (VH), fibrinoid content (PAS), and structural proteins (actin, myosin) of uterine arteries in ACE2 KO and C57Bl/6 mice. PAS, periodic acid Schiff stain; PSR, Picrosirius red stain; VH, Verhoeff's stain. Data are means ± SE. *P < 0.05 vs. C57Bl/6. Magnification ×40.

Fig. 4.

Analysis of uterine artery hemodynamics and trophoblast invasion in ACE2 KO and C57Bl/6 mice at day 14 of gestation. A: pulsed wave Doppler image and representative section of peak systolic (V_max) and minimum diastolic (V_min) velocities and analysis of the data in C57Bl/6 and ACE2 KO mice. Data are means ± SE; n = 4–6. B: distribution of cytokeratin-positive cells in the decidua (D) and mesometrium (M) of C57Bl/6 and ACE2 KO mice at ×5 and ×20 (inset) magnification; n = 3–4. L, labyrinth; JZ, junctional zone. Arrows indicate decidual arteries that are shown on the ×20 inset image.

Fig. 5.

Expression of hypoxia markers in the placenta of C57Bl/6 and ACE2 KO mice. A and B: pimonidazole staining and analysis. C and D: hypoxia-inducible factor-2α (HIF-2α) staining and analysis. Data are means ± SE; n = 4–5. *P < 0.05 vs. C57Bl/6. Magnification: A, ×5; C, ×20.

Fig. 6.

Analysis of umbilical artery velocities and the resistance index of C57Bl/6 and ACE2 KO mice at midgestation. A: representative wave forms of V_max and V_min points of umbilical artery velocity measurement. Arrows indicate V_max and V_min of the umbilical artery velocity wave. B: average measurements of V_max and V_min and the calculated resistance index. Data are means ± SE; n = 8–9. *P < 0.05 vs. C57Bl/6.