Estrogen receptor-α and estrogen receptor-β in the uterine vascular endothelium during pregnancy: functional implications for regulating uterine blood flow

Abstract

The steroid hormone estrogen and its classical estrogen receptors (ERs), ER-α and ER-β, have been shown to be partly responsible for the short- and long-term uterine endothelial adaptations during pregnancy. The ER-subtype molecular and structural differences coupled with the differential effects of estrogen in target cells and tissues suggest a substantial functional heterogeneity of the ERs in estrogen signaling. In this review we discuss (1) the role of estrogen and ERs in cardiovascular adaptations during pregnancy, (2) in vivo and in vitro expression of ERs in uterine artery endothelium during the ovarian cycle and pregnancy, contrasting reproductive and nonreproductive arterial endothelia, (3) the structural basis for functional diversity of the ERs and estrogen subtype selectivity, (4) the role of estrogen and ERs on genomic responses of uterine artery endothelial cells, and (5) the role of estrogen and ERs on nongenomic responses in uterine artery endothelia. These topics integrate current knowledge of this very rapidly expanding scientific field with diverse interpretations and hypotheses regarding the estrogenic effects that are mediated by either or both ERs and their relationship with vasodilatory and angiogenic vascular adaptations required for modulating the dramatic physiological rises in uteroplacental perfusion observed during normal pregnancy.

Figure 1

(A) Location of estrogen receptor (ER)-α and (B) ER-β mRNAs in ovine uterine arteries. Uterine arteries from pregnant sheep were fixed and paraffin embedded, and whole uterine artery sections (5 µm) were cut. Localization of ER-α and ER-β mRNAs was determined by in situ hybridization using S-labeled sense (control) or antisense riboprobes synthesized from a specific ovine ER-α cDNA or bovine ER-β cDNA. Silver grains shown in darkfield images represent positive mRNA labeling. Images were taken representative of n=7 at 10× magnification. BL, basal lamina; EC, endothelial cell; L, lumen; VSM, vascular smooth muscle. (With permission from Byers MJ, et al. J Physiol 2005;565:85–99.)

Figure 2

Immunohistochemical analysis of both (A) estrogen receptor (ER)-α and (B) ER-β (B) in uterine artery (UA) of pregnant ewes. Ovarian tissue from the same ewes was used as the positive control for ER-α and ER-β showing abundant expression of both ER-α and ER-β protein in the granulosa cells, but not the oocyte. Corresponding concentrations of rabbit and mouse immunoglobulin Gs served as nonspecific binding controls. E, endothelium; GC, granulosa cells; O, oocyte; SM, smooth muscle. Bar = 100 µm. (With permission from Liao WX, et al. Biol Reprod 2005;72:530–537.)

Figure 3

(A) Estrogen receptor (ER)-α and (B) ER-β protein expression in reproductive versus nonreproductive arterial endothelia. Western blot analysis was performed to evaluate the relative levels of ER-α and ER-β protein in luteal, follicular, and pregnant sheep reproductive and nonreproductive endothelia. Samples from luteal, follicular, and pregnant sheep are expressed as fold of the average of all luteal samples within an artery type, run on the same Western blots. Expression of PAendo is given as fold of the average luteal UAendo was also run on the same blot. Treatment groups: luteal: uterine (n = 12), mammary (n = 5), omental (n = 7), renal (n = 6), and coronary (n = 7). Follicular: uterine (n = 8), mammary (n = 5), omental (n = 8), renal (n = 6), and coronary (n = 8). Pregnant: uterine (n = 12), mammary (n = 6), placental (n = 8) omental (n = 8), renal (n = 8), and coronary (n = 8). Data are means plus or minus standard error of the mean. Means with different letters are statistically different (p < 0.05) within a tissue preparation. For ER-α UAendo: Lut < Fol (p < 0.05), For ER-β UAendo: Lut < Fol (p < 0.05) and Lut < Preg (p < 0.001); MAendo: Lut < Preg (p < 0.01); CAendo: Lut < Preg (p < 0.05). ^*For ER-α: PAendo < Luteal UAendo but for ER-β PAendo > Luteal UAendo (p < 0.05). (With permission from Byers MJ, et al. J Physiol 2005;565:85–99.)

Figure 4

Western immunoblot analysis of (A) estrogen receptor (ER)-α, (B) ER-β, and (C) β-actin in the protein extracts of uterine artery (UA), uterine artery endothelium (UAE), uterine artery endothelial cell (UAEC), and ovary from pregnant ewes. (D) A diagram representing the truncated form of ER-β2 that results from the splicing deletion of exon 5 shown in (B). The shadowed box represents the amino acid sequences encoded by different reading frame. aa, amino acid. Bands marked with asterisk may indicate additional truncated forms of ER-β. (With permission from Liao WX, et al. Biol Reprod 2005;72:530– 537.)

Figure 5

Concentration-dependent cell proliferation responses of nonpregnant uterine artery endothelial cells (NP-UAECs) and pregnant (P)-UAECs to E₂β. A biphasic proliferative response was observed in P-UAECs in response to E₂β compared with control with maximum responses at a physiological concentration of 0.1 nmol/L (two-way analysis of variance; pregnancy times concentration effect; E₂β, F_{4, 40}= 8.16, p < 0.0001. NP-UAECs did not respond to E₂β. Asterisk indicates increase (p < 0.05; n= 6) in P-UAEC proliferation compared with both the respective NP-UAEC (n = 7) group and untreated control. (With permission from Jobe SO, et al. Hypertension 2010;55:1005–1011.)

Figure 6

The effects of 1 µmol/L of ICI, MPP, and PHTPP on pregnant uterine artery endothelial cell (P-UAEC) proliferative responses to 0.1 nmol/L of E₂β. ICI and PHTPP, but not MPP, abrogated the response of P-UAECs to E₂β (two-way analysis of variance; antagonist times group effect; F_5,60 = 25.272, p < 0.001). Asterisks indicate increase (p < 0.05, n = 6) in P-UAEC proliferation compared with untreated control; τ indicates inhibition (p < 0.05) of P-UAEC proliferation with ICI and PHTPP. (With permission from Jobe SO, et al. Hypertension 2010;55:1005–1011.)

Figure 7

Concentration-dependent effects of (A) estrogen receptor (ER)-α agonist PPT, (B) ER-β agonist DPN, and (C) their combination on cell proliferation responses of P-UAECs. Blockade of ER-β with PHTPP (1 µmol/L) before treatment with ER-β agonist DNP is also shown in (C). ^* Increase (p < 0.05; n = 7) in P-UAEC proliferation compared with untreated controls. λ indicates a difference (p < 0.05) in P-UAEC proliferation in response to DPN or the combination of DNP and PPT compared with E₂β-only responses; τ inhibition (p < 0.05) of P-UAEC proliferation with PHTPP. (With permission from Jobe SO, et al. Hypertension 2010;55:1005–1011.)

Figure 8

Effect of estrogen receptor (ER)-α antibody on the nitric oxide synthase (NOS) activity response to E₂β. Isolated endothelial plasma membrane incubations were performed over 15 minutes in the absence (basal) or presence of E₂β with or without antibody to ER-α (TE111) or unrelated immunoglobulin G (IgG) added. Different letters are significantly different (p < 0.05). (Adapted from Chambliss KL, et al. Circ Res 2000;87:e44–e52.)

Figure 9

Effect of ICI 182,780 (10 nM) and THC (1 nM), the ER-β antagonist, on E₂β-mediated endothelial nitric oxide synthase (eNOS) activation in isolated endothelial cell caveolae membranes. ³H-L-arginine conversion to ³H-L citrulline was measured over 60 minutes. In caveolae, stimulated NOS activity ranged from 0.5 to 0.9 pmol citrulline per milligram protein^* min in separate studies. Different letters are significantly different (p < 0.05). Adapted from Chambliss KL, et al. Mol Endocrinol 2002;16:938–946.)