Endothelial PPARγ (Peroxisome Proliferator-Activated Receptor-γ) Protects From Angiotensin II-Induced Endothelial Dysfunction in Adult Offspring Born From Pregnancies Complicated by Hypertension

Abstract

Preeclampsia is a hypertensive disorder of pregnancy associated with vascular dysfunction and cardiovascular risk to offspring. We hypothesize that endothelial PPARγ (peroxisome proliferator-activated receptor-γ) provides cardiovascular protection in offspring from pregnancies complicated by hypertension. C57BL/6J dams were bred with E-V290M sires, which express a dominant-negative allele of PPARγ selectively in the endothelium. Arginine vasopressin was infused throughout gestation. Vasopressin elevated maternal blood pressure at gestational day 14 to 15 and urinary protein at day 17 consistent. Systolic blood pressure and vasodilation responses to acetylcholine were similar in vasopressin-exposed offspring compared to offspring from control pregnancies. We treated offspring with a subpressor dose of angiotensin II to test if hypertension during pregnancy predisposes offspring to hypertension. Male and female angiotensin II-treated E-V290M offspring from vasopressin-exposed but not control pregnancy exhibited significant impairment in acetylcholine-induced relaxation in carotid artery. Endothelial dysfunction in angiotensin II-treated E-V290M vasopressin-exposed offspring was attenuated by tempol, an effect which was more prominent in male offspring. Nrf2 (nuclear factor-E2-related factor) protein levels were significantly elevated in aorta from male E-V290M offspring, but not female offspring compared to controls. Blockade of ROCK (Rho-kinase) signaling and incubation with a ROCK2-specific inhibitor improved endothelial function in both male and female E-V290M offspring from vasopressin-exposed pregnancy. Our data suggest that interference with endothelial PPARγ in offspring from vasopressin-exposed pregnancies increases the risk for endothelial dysfunction on exposure to a cardiovascular stressor in adulthood. This implies that endothelial PPARγ provides protection to cardiovascular stressors in offspring of a pregnancy complicated by hypertension and perhaps in preeclampsia.

Keywords: angiotensin II; blood pressure; endothelium; preeclampsia; pregnancy; vasodilation.

Figure 1.. Experimental Design and Preeclamptic Phenotypes in the Mother

A) Schematic of the experimental design. Heterozygous E-V290M sires were bred with NT dams infused with either SAL (vehicle) or AVP. Offspring could either be wild-type (NT) or E-V290M. B) Longitudinal timeline of the study. Phase I is the gestational phase (in number of days) and Phase II is the life of offspring (in weeks). C-D) Preeclamptic phenotypes in the mother. C) Systolic blood pressure (SBP) was measured using tail-cuff plethysmography in SAL and AVP infused pregnant mice until gestational day (GD) 15. Data are presented as mean±SEM and analyzed by two-way repeated measures ANOVA, *P<0.05 vs. SAL pregnant. D) Urinary protein levels expressed as mg/mL (left) or mg/day (right) measured at baseline (base) and after infusion (GD17) of SAL or AVP in pregnant mice (Before SAL, n=6; GD17 SAL, n=9; before AVP, n=7; GD 17 AVP, n=20). Data are presented as mean±SEM and analyzed by two-way ANOVA. *P<0.05 vs. baseline.

Figure 2.. Ang-II Sensitivity in Adult Offspring

A) Systolic blood pressure measured using tail-cuff plethysmography in Ang-II-treated adult male (left) and female (right) NT and E-V290M offspring born from SAL or AVP-infused pregnancies. B) Cumulative concentration response curves for ACh in carotid arteries from Ang-II-treated adult male (left) and female (right) NT and E-V290M offspring born from SAL or AVP-infused pregnancies. C) 4-parameter non-linear curve fitting was used to calculate EC₅₀ and maximum responses for all relaxation curves. Baseline values were constrained to zero. All data are presented as mean±SEM analyzed by 2-way RM ANOVA with Tukey’s multiple comparison test. *P<0.05 vs E-V290M SAL, ^#P<0.05 vs NT AVP. D) Cumulative concentration response curves for SNP in carotid arteries from Ang-II-treated adult male (left) and female (right) NT and E-V290M offspring born from SAL or AVP-infused pregnancies. Data are presented as mean±SEM analyzed by two-way ANOVA with repeated measures.

Figure 3.. Endothelial Function in Adult Offspring: Role of ROS

A-B) Cumulative concentration response curves for ACh in carotid arteries with and without tempol (1mmol/L, 30 mins) in male (A) and female (B) offspring from the indicated pregnancy. S, saline; T, Tempol. Data are presented as mean±SEM analyzed by two-way ANOVA with repeated measures. *P<0.05 vs. E-V290M AVP + Tempol and Saline + Tempol. C-D) Representative western blots showing expression levels of Nrf2 and NQO1 in the aorta and its quantification from Ang-II-treated adult male (C) and female (D) NT and E-V290M offspring born from SAL or AVP-infused pregnancies (n=5–6 per group). Actual size markers transferred from the blots are shown. Data from the entire experiment are shown in the graphs. Data are presented as mean±SEM analyzed by two-way ANOVA. *P<0.05 vs. E-V290M SAL, ^$P<0.05 vs. NT AVP.

Figure 4.. Endothelial Function in Adult Offspring: Role of Rho-kinase

Cumulative concentration response curves for ACh in carotid arteries from adult male (A) and female (B) with and without ROCK inhibitor, Y-27632 (1 μmol/L, 30 min). Data are presented as mean±SEM analyzed by two-way ANOVA with repeated measures. *P<0.05 vs. E-V290M AVP + Y-27632.

Figure 5.. Protein expression RhoA-Rho kinase signaling pathway

Representative western blots showing expression levels of total RhoA, ROCK1, ROCK2 and P-MYPT in the aorta and its quantification from Ang-II-treated adult male (A) and female (B) NT and E-V290M offspring born from SAL or AVP-infused pregnancies (n=6 per group). Actual size markers transferred from the blots are shown. Data are presented as mean±SEM analyzed by two-way ANOVA. *P<0.05 vs. E-V290M SAL, ^$P<0.05 vs. NT AVP.

Figure 6.. Endothelial Function in Adult Offspring: Attenuation by ROCK2-specific inhibitor

Cumulative concentration response curves for ACh in carotid arteries from adult male (A) and female (B) with and without ROCK2-specific inhibitor, SLX-2119. Data are presented as mean±SEM analyzed by two-way ANOVA with repeated measures. *P<0.05 vs. E-V290M AVP + SLX-2119.