17β-Estradiol attenuates hypoxic pulmonary hypertension via estrogen receptor-mediated effects

Abstract

Rationale: 17β-Estradiol (E2) attenuates hypoxic pulmonary vasoconstriction and hypoxic pulmonary hypertension (HPH) through an unknown mechanism that may involve estrogen receptors (ER) or E2 conversion to catecholestradiols and methoxyestradiols with previously unrecognized effects on cardiopulmonary vascular remodeling.

Objectives: To determine the mechanism by which E2 exerts protective effects in HPH.

Methods: Male rats were exposed to hypobaric hypoxia while treated with E2 (75 μg/kg/d) or vehicle. Subgroups were cotreated with pharmacologic ER-antagonist or with inhibitors of E2-metabolite conversion. Complementary studies were performed in rats cotreated with selective ERα- or ERβ-antagonist. Hemodynamic and pulmonary artery (PA) and right ventricular (RV) remodeling parameters, including cell proliferation, cell cycle, and autophagy, were measured in vivo and in cultured primary rat PA endothelial cells.

Measurements and main results: E2 significantly attenuated HPH endpoints. Hypoxia increased ERβ but not ERα lung vascular expression. Co-treatment with nonselective ER inhibitor or ERα-specific antagonist rendered hypoxic animals resistant to the beneficial effects of E2 on cardiopulmonary hemodynamics, whereas ERα- and ERβ-specific antagonists opposed the remodeling effects of E2. In contrast, inhibition of E2-metabolite conversion did not abolish E2 protection. E2-treated hypoxic animals exhibited reduced ERK1/2 activation and increased expression of cell-cycle inhibitor p27(Kip1) in lungs and RV, with up-regulation of lung autophagy. E2-induced signaling was recapitulated in hypoxic but not normoxic endothelial cells, and was associated with decreased vascular endothelial growth factor secretion and cell proliferation.

Conclusions: E2 attenuates hemodynamic and remodeling parameters in HPH in an ER-dependent manner, through direct antiproliferative mechanisms on vascular cells, which may provide novel nonhormonal therapeutic targets for HPH.

Figure 1.

Treatment with 17β-estradiol (E2) conversion inhibitors does not attenuate E2 effects on hypoxia-induced pulmonary hypertension. (A) Right ventricular mass (RV/[LV+S]), (B) right ventricular systolic pressure (RVSP), (C) cardiac output (CO), (D) RVSP/CO, and (E) hematocrit (Hct) levels in male Sprague-Dawley rats exposed to normoxia (Fi_O2 21%) or chronic hypobaric hypoxia (P_atm = 362 mm Hg; equivalent to 10% Fi_O2; 2 wk). Rats were either untreated; treated with E2 alone (75 μg/kg/d via subcutaneously implanted osmotic minipumps for 1 wk before and throughout hypoxia exposure); or cotreated with E2 and the catechol O-methyltransferase inhibitor OR-486 (1.5 mg/kg) or the CYP450 inhibitor 1-aminobenzotriazole (ABT; 50 mg/kg). Additional animals were treated with OR-486 or ABT alone. OR-486 and ABT were administered daily subcutaneously for 1 week before and for the entire 2 weeks of hypoxia exposure. Values are mean ± SEM (^‡P < 0.001, ^††P < 0.01 vs. normoxia group; **P < 0.001, *P < 0.01, ^#P < 0.05 for all other comparisons; A and E, n = 13 for hypoxia E2, n = 6–12 per group for all other groups; B–D, n = 12 for hypoxia E2, n = 5–9 per group for all other groups).

Figure 2.

Estrogen receptor blockade with ICI182780 (fulvestrant [ICI]) attenuates protective effects of 17β-estradiol (E2) in hypoxia-induced pulmonary hypertension. (A) Right ventricular mass (RV/[LV+S]), (B) right ventricular systolic pressure (RVSP), (C) cardiac output (CO), (D) RVSP/CO, and (E) hematocrit (Hct) levels in normoxic and hypoxic (P_atm = 362 mm Hg; 10% Fi_O2; 2 wk) male rats treated with E2 (75 μg/kg/d; 1 wk before and during hypoxia) with or without ICI (3 mg/kg/d subcutaneously). Values are mean ± SEM (**P < 0.001, *P < 0.01, ^#P < 0.05; A and E, n = 13 for hypoxia E2, n = 6–12 per group for all other groups; B–D, n = 12 for hypoxia E2, n = 5–9 per group for all other groups).

Figure 3.

17β-Estradiol (E2) attenuation of hypoxia-induced pulmonary artery (PA) muscularization is less pronounced after estrogen receptor (ER) blockade. (A) Representative images of α-smooth muscle actin (α-SMA) lung immunohistochemistry showing PAs associated with alveolar ducts (AD) of male rats exposed to 2 weeks of normoxia or hypobaric hypoxia. Hypoxia-exposed animals were either untreated, or treated with E2, E2 + ICI182780 (ICI), E2 + OR-486 (OR), or E2 + 1-aminobenzotriazole (ABT). Size bars = 100 μm. Note that hypoxia increased PA muscularization, resulting in homogenous thickening of the smooth muscle cell layer (arrowheads), whereas PAs of E2-treated rats with hypoxic pulmonary hypertension exhibited less complete muscularization (arrows). The E2-induced decrease in muscularization was attenuated after ER-antagonist (ICI) co-treatment, whereas treatment with E2 conversion inhibitors (OR-486 and 1-ABT) did not have a significant effect. (B) Quantification of PA muscularization. Alveolar duct–associated PAs (<200 μm) were divided into nonmuscularized (α-SMA staining <25% of vessel circumference), partially muscularized (α-SMA staining 25–74% of vessel circumference), or fully muscularized (α-SMA staining ≥75% of vessel circumference) vessels. Percentages of nonmuscularized (white bars), partially muscularized (light gray bars), or fully muscularized vessels (dark gray bars) were calculated. E2 treatment increased the percentage of nonmuscularized vessels compared with the untreated hypoxia group. In contrast, ER blockade in the presence of E2 treatment was associated with a significant increase in the percentage of fully muscularized vessels compared with E2 alone. Values are mean ± SEM (^††P < 0.01, ^†P < 0.05 vs. untreated normoxia group, ^#P < 0.05; n = 4–7 per group).

Figure 4.

17β-Estradiol (E2) decreases hypoxia-induced right ventricular (RV) remodeling. (A) Representative confocal microscopic images of RV sections from normoxic and hypoxic pulmonary hypertension rats. Hypoxic pulmonary hypertension animals were either untreated, or treated with E2 or E2 + ICI182780 (ICI). Glycocalyx is stained in green (wheat germ agglutinin conjugated to Oregon Green 488). Green staining therefore indicates cell membranes of myocytes or capillary endothelial cells. RV myocyte nuclei are stained in blue (DAPI). Capillaries (arrows) were identified by blood autofluorescence surrounded by glycocalyx staining of the endothelial cell membrane. Myocytes (arrowheads) were identified by size and shape (indicated by glycocalyx staining of the cell membrane), in conjunction with myoglobin autofluorescence surrounded by membrane glycocalyx staining. Size bars = 50 μm. Note that hypoxia increased RV capillarization, whereas E2 treatment attenuated it, but to a lesser extent in rats cotreated with estrogen receptor antagonist. (B) Quantification of capillary density, expressed as the ratio of capillaries to the number of myocytes per high-power field. Note that estrogen receptor blockade decreased E2 effect on hypoxia-induced RV remodeling. Values are mean ± SEM (^#P < 0.05, **P < 0.001; n.s. = not statistically significant; n = 4 animals per group).

Figure 5.

Estrogen receptor (ER)-α blockade attenuates 17β-estradiol (E2) protection in hypoxic pulmonary hypertension. (A) Right ventricular mass (RV/[LV+S]), (B) right ventricular systolic pressure (RVSP), (C) cardiac output (CO), (D) RVSP/CO, and (E) hematocrit (Hct) levels in normoxic and hypoxic male rats treated with E2 alone (75 μg/kg/d) or with E2 in combination with the selective ERα antagonist MPP (850 μg/kg/d) or the selective ERβ antagonist PHTPP (850 μg/kg/d). All drugs were started 1 week before hypoxia and continued throughout the entire 2-week hypoxia exposure. E2 was administered via subcutaneous osmotic minipumps; MPP and PHTPP were injected subcutaneously once per day. Values are mean ± SEM (**P < 0.001; n = 4–12 per group).

Figure 6.

Selective estrogen receptor (ER)α- or ERβ-blockade attenuates protective 17β-estradiol (E2) effects on pulmonary artery (PA) remodeling (A), but does not significantly affect right ventricular (RV) capillarization (B) in E2-treated hypoxic pulmonary hypertension rats. (A) Representative images and quantification of muscularization of alveolar duct (AD)–associated PAs (<200 μm). Hypoxia-exposed animals were either untreated, or treated with E2, E2 + MPP (ERα antagonist; 850 μg/kg/d), or E2 + PHTPP (ERβ antagonist; 850 μg/kg/d). Immunohistochemistry for α-smooth muscle actin was performed, and degree of PA muscularization (nonmuscularized, partially muscularized, or fully muscularized) was determined as outlined in Figure 3. Size bars = 100 μm. Note that E2-associated decrease in hypoxia-induced PA muscularization (evidenced by areas of less complete muscularization [arrows]) was attenuated after co-treatment with ERα- or ERβ-antagonist, resulting in more complete PA muscularization (arrowheads). (B) Representative images and quantification of RV capillarization in untreated hypoxic pulmonary hypertension rats, or hypoxic rats treated with E2, E2 + MPP, or E2 + PHTPP. RV capillarization was determined as outlined in Figure 4. Arrows indicate representative capillaries; arrowheads indicate representative myocytes. Size bars = 50 μm. Note that neither coadministration of MPP nor PHTPP attenuated E2 effects on capillary/myocyte ratio. Values are mean ± SEM (^†P < 0.05, ^††P < 0.01 vs. hypoxia + E2 nonmuscularized; ^#P < 0.05, ^‡P = 0.07 vs. hypoxia + E2 fully muscularized; n = 4–6 animals per group).

Figure 7.

17β-Estradiol (E2) decreases ERK1/2 activation in hypoxic pulmonary hypertension lungs and right ventricles (RVs) in an estrogen receptor (ER)–dependent manner. Activation of ERK1/2 in lungs (A) and RVs (B) of rats exposed to normoxia or hypobaric hypoxia (2 wk). Hypoxic rats were either untreated, treated with E2 alone, or cotreated with E2 and ICI182780 (ICI). Representative Western blots of phospho-ERK1/2, total ERK1/2, and vinculin (for RV) are shown in the upper panels. Bar graphs represent relative increase in phospho-ERK1/2 to total ERK1/2 measured by densitometry (mean ± SEM; ^#P < 0.05; n.s. = not statistically significant). (A) n = 6, (B) n = 4–5.

Figure 8.

17β-Estradiol (E2) increases expression of cell cycle inhibitor p27^Kip1 (A and B) and autophagy marker LC3-II (C) in hypoxic pulmonary hypertension rats. Expression of p27^Kip1 and LC3-II are demonstrated in lungs (A and C) and right ventricles (RVs) (B) of rats exposed to normoxia or hypobaric hypoxia (2 wk) and treated with E2. Representative Western blots of p27^Kip1 and LC3-II are shown in the upper panels. Bar graphs represent relative increase in p27^Kip1 and LC3-II, respectively, to vinculin measured by densitometry (mean ± SEM; normox = untreated normoxia group; con = untreated hypoxia control; ^#P < 0.05; *P < 0.01; n.s. = not statistically significant). (A) n = 4, (B) n = 5, (C) n = 3–5.

Figure 9.

Hypoxic pulmonary hypertension is associated with increased lung estrogen receptor (ER)β-expression. Male Sprague-Dawley rats were exposed to 2 weeks of hypobaric hypoxia (P_atm = 362 mm Hg; equivalent to 10% Fi_O2). Lungs were harvested, and immunohistochemistry (IHC) for ERβ (A) or Western blotting for ERα (B) was performed. ERα was not detectable by IHC. Representative IHC images (for ERβ; A) and Western blots (for ERα and vinculin loading control; B) are shown. Note the increase in ERβ-positive cells (arrows) in hypoxic pulmonary arteries (PA) at the level of terminal bronchioles (TB) or alveolar ducts (AD). Of note, positive staining for ERβ mainly occurred in PA endothelial cells, whereas there was no significant staining of PA smooth muscle cells. Size bars = 50 μm.

Figure 10.

17β-Estradiol (E2) decreases ERK1/2 activation and increases cell cycle inhibitor p27^Kip1 and autophagy parameter LC3-II in hypoxic, but not normoxic primary rat pulmonary artery cells (RPAECs). ERK1/2 activation (A–C), p27^Kip1 expression (D and E), and LC3-II expression (F and G) in RPAECs grown at 21% or 1% O₂ for 48 hours and either untreated (con) or treated with E2 or vehicle (veh). In (B) and (C), E2-treated cells were also cotreated with nonselective (ICI182780 [ICI]) or selective estrogen receptor (ER)α- (MPP) and ERβ-antagonists (PHTPP), respectively. Representative Western blots are shown in the upper panels. Bar graphs represent relative increase in phospho-ERK1/2 to total ERK1/2, in p27^Kip1 to vinculin, and in LC3-II to actin, all measured by densitometry. Values (mean ± SEM) are expressed as fold change compared with normoxic or hypoxic control group, respectively, except for C, where values are expressed as fold change compared with hypoxic E2 group (A and B, D–G, ^#P < 0.05, **P < 0.001, ***P < 0.0001; C, ^#P < 0.05, *P < 0.01 vs. E2; n = 3 independent experiments).

Figure 11.

17β-Estradiol (E2) decreases primary rat pulmonary artery cell (RPAEC) proliferation in hypoxia. (A) Bromodeoxyuridine (BrdU) uptake in RPAECs grown at 21% or 1% O₂ for 48 hours in the presence of E2, E2 ± ICI182780 (ICI), or E2 vehicle (veh). (B) Viable RPAECs (assessed by trypan blue uptake) exposed to 21% (white bars) or 1% O₂ (black bars) for 48 hours and treated with E2 or vehicle. Number of viable cells in E2- or vehicle-treated normoxia and hypoxia groups is expressed as fold change from their untreated control group, respectively. Values are mean ± SEM (con = control; OD = optical density; ^#P < 0.05, **P < 0.001, ***P < 0.0001).