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. 2015 Nov 15;309(10):H1679-96.
doi: 10.1152/ajpheart.00532.2015. Epub 2015 Sep 25.

Adaptive increases in expression and vasodilator activity of estrogen receptor subtypes in a blood vessel-specific pattern during pregnancy

Karina M Mata  1 , Wei Li  1 , Ossama M Reslan  1 , Waleed T Siddiqui  1 , Lauren A Opsasnick  1 , Raouf A Khalil  2
Affiliations

Adaptive increases in expression and vasodilator activity of estrogen receptor subtypes in a blood vessel-specific pattern during pregnancy

Karina M Mata et al. Am J Physiol Heart Circ Physiol. .

Abstract

Normal pregnancy is associated with adaptive hemodynamic, hormonal, and vascular changes, and estrogen (E2) may promote vasodilation during pregnancy; however, the specific E2 receptor (ER) subtype, post-ER signaling mechanism, and vascular bed involved are unclear. We tested whether pregnancy-associated vascular adaptations involve changes in the expression/distribution/activity of distinct ER subtypes in a blood vessel-specific manner. Blood pressure (BP) and plasma E2 were measured in virgin and pregnant (day 19) rats, and the thoracic aorta, carotid artery, mesenteric artery, and renal artery were isolated for measurements of ERα, ERβ, and G protein-coupled receptor 30 [G protein-coupled ER (GPER)] expression and tissue distribution in parallel with relaxation responses to E2 (all ERs) and the specific ER agonist 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)-tris-phenol (PPT; ERα), diarylpropionitrile (DPN; ERβ), and G1 (GPER). BP was slightly lower and plasma E2 was higher in pregnant versus virgin rats. Western blots revealed increased ERα and ERβ in the aorta and mesenteric artery and GPER in the aorta of pregnant versus virgin rats. Immunohistochemistry revealed that the increases in ERs were mainly in the intima and media. In phenylephrine-precontracted vessels, E2 and PPT caused relaxation that was greater in the aorta and mesenteric artery but similar in the carotid and renal artery of pregnant versus virgin rats. DPN- and G1-induced relaxation was greater in the mesenteric and renal artery than in the aorta and carotid artery, and aortic relaxation to G1 was greater in pregnant versus virgin rats. The nitric oxide synthase inhibitor N(ω)-nitro-l-arginine methyl ester with or without the cyclooxygenase inhibitor indomethacin with or without the EDHF blocker tetraethylammonium or endothelium removal reduced E2, PPT, and G1-induced relaxation in the aorta of pregnant rats, suggesting an endothelium-dependent mechanism, but did not affect E2-, PPT-, DPN-, or G1-induced relaxation in other vessels, suggesting endothelium-independent mechanisms. E2, PPT, DPN, and G1 caused relaxation of Ca(2+) entry-dependent KCl contraction, and the effect of PPT was greater in the mesenteric artery of pregnant versus virgin rats. Thus, during pregnancy, an increase in ERα expression in endothelial and vascular smooth muscle layers of the aorta and mesenteric artery is associated with increased ERα-mediated relaxation via endothelium-derived vasodilators and inhibition of Ca(2+) entry into vascular smooth muscle, supporting a role of aortic and mesenteric arterial ERα in pregnancy-associated vasodilation. GPER may contribute to aortic relaxation while enhanced ERβ expression could mediate other genomic vascular effects during pregnancy.

Keywords: calcium; endothelium; estrogen; nitric oxide; vascular smooth muscle.

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Figures

Fig. 1.
Fig. 1.
Blood pressure (BP), plasma estrogen/estradiol (E2), body weight, and tissue weight in virgin versus pregnant (Preg) rats. BP was recorded (A) and plasma E2 levels were measured by ELISA (B) in virgin and pregnant (day 19) rats. Body weight with and without (w/o) the uterus (C) and uterus weight (D) were compared. Arterial tissue weight (E) and kidney weight (F) were measured and presented as a percentage of body weight without the uterus. Bar graphs represent means ± SE; n = 5–6 rats/group. *P < 0.05, pregnant vs. virgin rats; #P < 0.05, without the uterus vs. with the uterus.
Fig. 2.
Fig. 2.
Protein amount and tissue distribution of estrogen receptor (ER)α in the aorta (A), carotid artery (C), mesenteric artery (M), and renal artery (R) as well as the uterus of virgin versus pregnant rats. Tissue homogenates of blood vessels (A) and the uterus (B) were prepared for Western blots using ERα antibody (1:1,000). ERα and G protein-coupled ER (GPER; shown in Fig. 4) were run on the same gel and therefore have the same actin control. Vascular tissue sections (C) and uterine sections (D) were stained with ERα antibody using immunohistochemistry, and the total amount and relative distribution of ERα brown immunostaining in different layers of the tissue wall were measured using ImageJ. Bar graphs represent means ± SE; n = 4–6 rats/group. *P < 0.05, pregnant vs. virgin rats; ‡significantly different (P < 0.05) from corresponding measurements in the aorta, carotid artery, mesenteric artery, and renal artery of virgin rats; #significantly different (P < 0.05) from corresponding measurements in the aorta, carotid artery, mesenteric artery, and renal artery of pregnant rats.
Fig. 3.
Fig. 3.
Protein amount and tissue distribution of ERβ in the aorta, carotid artery, mesenteric artery, and renal artery as well as the uterus of virgin versus pregnant rats. Tissue homogenates of blood vessels (A) and the uterus (B) were prepared for Western blots using ERβ antibody (1:1,000). Vascular tissue sections (C) and uterine sections (D) were stained with ERβ antibody using immunohistochemistry, and the total amount and relative distribution of ERβ brown immunostaining in different layers of the tissue wall were measured using ImageJ. Bar graphs represent means ± SE; n = 4–6 rats/group. *P < 0.05, pregnant vs. virgin rats; ‡significantly different (P < 0.05) from corresponding measurements in the aorta, carotid artery, mesenteric artery, and renal artery of virgin rats; #significantly different (P < 0.05) from corresponding measurements in the aorta, carotid artery, mesenteric artery, and renal artery of pregnant rats.
Fig. 4.
Fig. 4.
Protein amount and tissue distribution of GPER in the aorta, carotid artery, mesenteric artery, and renal artery as well as the uterus of virgin versus pregnant rats. Tissue homogenates of blood vessels (A) and the uterus (B) were prepared for Western blots using GPER antibody (1:1,000). GPER and ERα (shown in Fig. 2) were run on the same gel and therefore have the same actin control. Vascular tissue sections (C) and uterine sections (D) were stained with GPER antibody using immunohistochemistry, and the total amount and relative distribution of GPER brown immunostaining in different layers of the tissue wall were measured using ImageJ. Bar graphs represent means ± SE; n = 4–6 rats/group. *P < 0.05, preg vs. virgin rats; ‡significantly different (P < 0.05) from corresponding measurements in the aorta, carotid artery, mesenteric artery, and renal artery of virgin rats; #significantly different (P < 0.05) from corresponding measurements in the aorta, carotid artery, mesenteric artery, and renal artery of pregnant rats.
Fig. 5.
Fig. 5.
Histology and morphometric analysis of the aorta, carotid artery, mesenteric artery, and renal artery (A and C) as well as the uterus (B and D) of virgin versus pregnant rats. Cryosections were prepared for hematoxylin and eosin staining, and tissue images were analyzed using ImageJ. Total tissue area, lumen area, whole wall area, and wall-to-lumen ratio were measured. Total wall thickness and the relative thickness of the different vascular layers (intima, media, and adventitia) and uterine layers (endometrium, myometrium, and perimetrium) were also measured. Bar graphs represent means ± SE; n = 4–6 rats/group. *P < 0.05, pregnant vs. virgin rats.
Fig. 6.
Fig. 6.
Differential effects of E2, 4,4′,4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)-tris-phenol (PPT), diarylpropionitrile (DPN), G1, and ACh on relaxation of the phenylephrine (Phe)-precontracted aorta (A), carotid artery (B), mesenteric artery (C), and renal artery (D) of virgin versus pregnant rats. Endothelium-intact segments of the thoracic aorta, carotid artery, mesenteric artery, and renal artery were precontracted with Phe. Increasing concentrations (10−12–10−5 M) of E2 (activator of all ERs), PPT (ERα agonist), DPN (ERβ agonist), or G1 (GPER agonist) were added, and the percent relaxation of Phe-induced contraction was measured. In parallel experiments, the effects of increasing concentrations (10−9–10−5 M) of ACh on the relaxation of Phe-precontracted vessels were measured. Data represent means ± SE; n = 8–10 rats/group. *P < 0.05, pregnant vs. virgin rats.
Fig. 7.
Fig. 7.
Effect of blockade of nitric oxide, prostglandins, and EDHF or endothelium removal (Endo) on the vascular relaxation effects of E2, PPT, DPN, and G1 in the aorta (A), carotid artery (B), mesenteric artery (C), and renal artery (D) of pregnant rats. Vascular segments from pregnant rats were either nontreated or pretreated with the nitric oxide inhibitor Nω-nitro-l-arginine methyl ester (l-NAME; 3 × 10−4 M), cyclooxygenase inhibitor indomethacin (Indo; 10−5 M), and the hyperpolarization blocker tetraethylammonium (TEA; 30 mM) for 15 min or endothelium denuded. Vascular segments were then precontracted with submaximal concentrations of Phe. Increasing concentrations (10−12–10−5 M) of E2, PPT, DPN, and G1 were then added, and the relaxation response was measured. Data represent means ± SE; n = 7–11 rats/group. *Significantly different (P < 0.05) from corresponding measurement in control nontreated intact vessels.
Fig. 8.
Fig. 8.
Differential effects of E2, PPT, DPN, and G1 on relaxation of the KCl-precontracted aorta (A), carotid artery (B), mesenteric artery (C), and renal artery (D) of virgin versus pregnant rats. Endothelium-denuded segments of the thoracic aorta, carotid artery, mesenteric artery, and renal artery were precontracted with high KCl (96 mM) depolarizing solution to induce a Ca2+-dependent contractile response in vascular smooth muscle. Increasing concentrations (10−12–10−5 M) of E2 (activator of all ERs), PPT (ERα agonist), DPN (ERβ agonist), or G1 (GPER agonist) were added, and the percent relaxation of KCl contraction was measured. Data represent means ± SE; n = 7–10 rats/group. *P < 0.05, pregnant vs. virgin rats.
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