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. 2011 Sep 15;184(6):715-23.
doi: 10.1164/rccm.201101-0078OC. Epub 2011 Jun 23.

Estrogen rescues preexisting severe pulmonary hypertension in rats

Soban Umar  1 , Andrea Iorga, Humann Matori, Rangarajan D Nadadur, Jingyuan Li, Federica Maltese, Arnoud van der Laarse, Mansoureh Eghbali
Affiliations

Estrogen rescues preexisting severe pulmonary hypertension in rats

Soban Umar et al. Am J Respir Crit Care Med. .

Abstract

Rationale: Pulmonary hypertension (PH) is characterized by progressive increase in pulmonary artery pressure leading to right ventricular (RV) hypertrophy, RV failure, and death. Current treatments only temporarily reduce severity of the disease, and an ideal therapy is still lacking.

Objectives: Estrogen pretreatment has been shown to attenuate development of PH. Because PH is not often diagnosed early, we examined if estrogen can rescue preexisting advanced PH.

Methods: PH was induced in male rats with monocrotaline (60 mg/kg). At Day 21, rats were either treated with 17-β estradiol or estrogen (E2, 42.5 μg/kg/d), estrogen receptor-β agonist (diarylpropionitrile, 850 μg/kg/d), or estrogen receptor α-agonist (4,4',4"-[4-Propyl-(1H)-pyrazole-1,3,5-triyl] trisphenol, 850 μg/kg/d) for 10 days or left untreated to develop RV failure. Serial echocardiography, cardiac catheterization, immunohistochemistry, Western blot, and real-time polymerase chain reaction were performed.

Measurements and main results: Estrogen therapy prevented progression of PH to RV failure and restored lung and RV structure and function. This restoration was maintained even after removal of estrogen at Day 30, resulting in 100% survival at Day 42. Estradiol treatment restored the loss of blood vessels in the lungs and RV. In the presence of angiogenesis inhibitor TNP-470 (30 mg/kg) or estrogen receptor-β antagonist (PHTPP, 850 μg/kg/d), estrogen failed to rescue PH. Estrogen receptor-β selective agonist was as effective as estrogen in rescuing PH.

Conclusions: Estrogen rescues preexisting severe PH in rats by restoring lung and RV structure and function that are maintained even after removal of estrogen. Estrogen-induced rescue of PH is associated with stimulation of cardiopulmonary neoangiogenesis, suppression of inflammation, fibrosis, and RV hypertrophy. Furthermore, estrogen rescue is likely mediated through estrogen receptor-β.

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Figures

Figure 1.
Figure 1.
Estrogen (E2) rescues severe pulmonary hypertension (PH). (A) Experimental protocol: male rats were injected with monocrotaline (MCT) or phosphate-buffered saline (PBS) at Day 0. The thick horizontal lines represent the length of each experimental group. At Day 21, animals were either killed (PH group), or left untreated to develop right ventricular (RV) failure (RVF group). Both E2 and E2-W groups received E2 only from Day 21 to Day 30. All of the rats in the E2 group were killed at Day 30, whereas the rats in the E2-W group were kept for another 12 days until Day 42 after E2 withdrawal at Day 30. (B) Survival plot. (C) RV systolic pressure (RVSP) and (D) RV ejection fraction (RVEF) at baseline (open circle) and after a single MCT/PBS injection in control mice (CTRL; black circle), PH (red circle), RVF (purple circle), and E2-treated (blue squares). For simplicity, RVSP and RVEF are only shown at the end of the experiment, except for the E2-treated group, for which these parameters are shown as a function of time. The black dotted lines represent the baseline levels. (E) Lung weight and (F) RV hypertrophy index in CTRL (black bar), PH (red bar), RVF (purple bar), E2 (dark blue bar) and E2-W (light blue bar). *P < 0.05 versus CTRL, **P < 0.001 versus CTRL; ††P < 0.001 versus PH; ^^P < 0.001 versus RVF (n = 6–8 rats per group). IVS = interventricular septum; LV = left ventricular wall.
Figure 2.
Figure 2.
Reversal of cardiopulmonary structure and function by estrogen (E2). (A) hematoxylin-eosin staining for lung arterioles and heart cross-sections in male rats. (B) Echocardiographic images of M-mode (upper panels) showing right ventricular (RV) end-diastolic diameter (EDD), left ventricular (LV) and interventricular septum (IVS), and pulse-wave Doppler (lower panels) in male rats. Yellow arrows show mid-systolic notch present in pulmonary hypertension (PH) and RV failure (RVF) only.
Figure 3.
Figure 3.
Reversal of lung inflammation and remodeling by estrogen (E2). (A) Lung sections of male rats stained for ED1 in control (CTRL), pulmonary hypertension (PH), and estrogen (E2) groups (upper panel) and at higher magnification of the respective fields (lower panel). Red arrows indicate the ED1-positive cells. (B) ED1-positive cells quantification per field in CTRL (black bar), PH (red bar), and E2 (blue bar). (C) Relative transcript expression of lung IL-6 in CTRL (black bar), PH (red bar), and E2 (blue bar), normalized to CTRL. (D) Representative immunoblots of lung lysates from CTRL, PH, and E2 labeled with anti–IL-6 and anti-vinculin antibodies. In this immunoblot and the subsequent immunoblots, all samples from CTRL, PH, and E2 groups were run on the same gel. Because we are only showing representative lanes from a total of three to five samples per group, some of the intervening lanes were not shown and are separated by a dotted line. (E) Western blot analysis of IL-6 protein in lung lysates normalized to vinculin in CTRL (black bar), PH (red bar), and E2 (blue bar). (F) Masson trichrome staining of lung sections in CTRL, PH, and E2; blue color indicates fibrosis. (G) Quantification of lung fibrosis showing percent lung fibrosis in CTRL, PH, and E2 groups. (H) Immunofluorescence labeling of pulmonary arterioles stained for smooth muscle actin (green) and (I) immunoperoxidase labeling of pulmonary arterioles stained with anti–smooth muscle actin antibody (brown) together with hematoxylin-stained nuclei (blue). (J) Bar graph for quantification of pulmonary arteriolar medial wall thickness in CTRL, PH, and E2 groups. *P < 0.05 versus CTRL, **P < 0.001 versus CTRL; †P < 0.05 versus PH, ††P < 0.001 versus PH (n = 4 animals per group).
Figure 4.
Figure 4.
Stimulation of pulmonary neoangiogenesis by estrogen (E2). (A) Single confocal images of lung sections of male rats immunostained for von Willebrand Factor (green, upper panels), overlay of von Willebrand factor and nuclei (stained red with TO PRO, middle panels), and at higher magnification of the respective fields (lower panels). (B) Quantification of vessels per high power field (HPF) in control (CTRL; black bar), pulmonary hypertension (PH; red bar), and E2 (blue bar). **P < 0.001 versus CTRL; ††P < 0.001 versus PH (n = 4 animals per group).
Figure 5.
Figure 5.
Stimulation of cardiac neoangiogenesis by estrogen (E2). (A–C) Single confocal images of right ventricular sections of male rats immunostained for CD31 (green, A), overlay of CD31 and WGA (red, B) and at higher display magnification (C). (D) Quantification of microvessels/cardiomyocyte in control (CTRL; black bar), pulmonary hypertension (PH; red), and E2 (blue). *P < 0.05 versus CTRL, **P < 0.001 versus CTRL; ††P < 0.001 versus PH (n = 4 animals per group).
Figure 6.
Figure 6.
Estrogen (E2) fails to rescue pulmonary hypertension (PH) in the presence of angiogenesis inhibitor. (A) Experimental protocol. (B) Right ventricular systolic pressure (RVSP) and (C) RV ejection fraction (RVEF) at the end point of each experiment, except for E2-treated groups, for which the data are presented as a function of time. The solid lines represent the baseline levels. (D–F) RVSP, RVEF, and RV hypertrophy index RV/(left ventricular[LV]+ interventricular septum [IVS]) for control (CTRL; open bar), PH (shaded gray bar), E2 (gray bar), and E2+TNP (solid bar) groups at Day 30 except for PH group, which was at Day 21. *P < 0.05 versus CTRL, **P < 0.001 versus CTRL; †P < 0.05 versus PH, ††P < 0.001 versus PH; #P < 0.05 versus E2, ##P < 0.001 versus E2 (n = 3–8 animals per group).
Figure 7.
Figure 7.
Selective estrogen receptor (ER)β agonist diarylpropionitrile (DPN) is as effective as estrogen (E2) in rescuing severe pulmonary hypertension (PH), and E2 fails to rescue PH in the presence of ERβ antagonist, PHTPP. (A) Experimental design. (B–E) Right ventricular systolic pressure (RVSP) (B), RV ejection fraction (RVEF) (C), lung weight (D), and RV hypertrophy index (E), at Day 30 except for PH, which was at Day 21 after monocrotaline (MCT) injection, for control (CTRL; open bar), PH (solid bar), E2 (gray bar), DPN (ERβ-agonist, vertical striped bar), PPT (ERα agonist, shaded gray bar), and E2+PHTPP (ERβ antagonist, horizontal striped bar) groups in male rats. *P < 0.05 versus CTRL, **P < 0.001 versus CTRL; ††P < 0.001 versus PH; #P < 0.05 versus E2, ##P < 0.001 versus E2; $P < 0.05 versus DPN, $$P < 0.001 versus DPN (n = 3–8 animals per group).
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