Oestrogen inhibition reverses pulmonary arterial hypertension and associated metabolic defects

Abstract

Increased oestrogen is a strong epidemiological risk factor for development of pulmonary arterial hypertension (PAH) in patients, associated with metabolic defects. In addition, oestrogens drive penetrance in mice carrying mutations in bone morphogenetic protein receptor type II (BMPR2), the cause of most heritable PAH. The goal of the present study was to determine whether inhibition of oestrogens was effective in the treatment of PAH in these mice.The oestrogen inhibitors fulvestrant and anastrozole were used in a prevention and treatment paradigm in BMPR2 mutant mice, and tamoxifen was used for treatment. In addition, BMPR2 mutant mice were crossed onto oestrogen receptor (ESR)1 and ESR2 knockout backgrounds to assess receptor specificity. Haemodynamic and metabolic outcomes were measured.Oestrogen inhibition both prevented and treated PAH in BMPR2 mutant mice. This was associated with reduction in metabolic defects including oxidised lipid formation, insulin resistance and rescue of peroxisome proliferator-activated receptor-γ and CD36. The effect was mediated primarily through ESR2, but partially through ESR1.Our data suggest that trials of oestrogen inhibition in human PAH are warranted, and may improve pulmonary vascular disease through amelioration of metabolic defects. Although fulvestrant and anastrozole were more effective than tamoxifen, tamoxifen may be useful in premenopausal females, because of a reduced risk of induction of menopause.

Figure 1 –

(A) Anastrozole & Fulvestrant (A+F) reduce uterine weights in mice (p<0.0001) as expected; by multiple ANOVA, R899X mutation did not have an effect on uterine weights. Each symbol is the measurement from one mouse. (B) Anastrozole & Fulvestrant delivered in osmotic pumps through the final four weeks of six weeks’ BMPR2^R899X transgene induction prevents development of elevated RVSP (p<0.0001 by multiple ANOVA, using transgene and drug as variables). Each symbol is a value from one mouse; symbols that are triangles also received the androgen MPA, which did not impact RVSP and so have been not been separated. Numbers of mice in each group are listed at the bottom of the plot; mice were age-matched, and controls were done contemporaneously for each group. (C) Cardiac outputs were not significantly changed by either Anastrozole & Fulvestrant treatment or Bmpr2 mutation. (D) Anastrozole & Fulvestrant nearly normalized the increase in muscularized vessels normally seen in BMPR2^R899X mice. By multiple ANOVA considering BMPR2 mutation and treatment as factors, BMPR2 mutation increases muscularized vessels (p=0.0004) while A+F decrease them, specifically in BMPR2 mutant mice (p=0.003). (E) Staining for oxidized lipids demonstrates increased oxidative stress in BMPR2^R899X mice, normalized with Anastrozole & Fulvestrant. (F) Ceramide (toxic lipid) staining (brown) is increased in lungs from BMPR2^R899X mice, and reduced by Anastrozole and Fulvestrant.

Figure 2 –

(A) Anastrozole & Fulvestrant but not Tamoxifen used as treatment reduces uterine weights in mice (p=0.002 for comparison to control, or 0.007 for comparison to vehicle). Each symbol is the measurement from one mouse; median and SEM are indicated. (B) Anastrozole & Fulvestrant delivered in osmotic pumps through the final four weeks of ten weeks’ BMPR2^R899X transgene induction reverses development of elevated RVSP (p=0.003 by multiple ANOVA, using transgene and drug as variables). Tamoxifen used the same way did not have a significant effect on RVSP but RVSP trended down. Each symbol is a value from one mouse. (C) Anastrozole & Fulvestrant used as treatment nearly normalized the increase in muscularized vessels normally seen in BMPR2^R899X mice; Tamoxifen had intermediate effect. By ANOVA, BMPR2 mutation increases muscularized vessels (p=0.0004) while A+F (p=0.0004) and Tamoxifen (p=0.02) both decrease them. (D) Ceramide (toxic lipid) staining (brown) is increased in lungs from BMPR2^R899X mice, and reduced by Anastrozole and Fulvestrant, but not by Tamoxifen.

Figure 3 –

(A) ESR1 but not ESR2 knockout reduces uterine weights in mice (p<0.0001) as expected[33]; by multiple ANOVA, R899X mutation did not have an effect on uterine weights. Each symbol is the measurement from one mouse; median and SEM are indicated. (B) ESR1 knockout partially and ESR2 knockout completely reduces RVSP in both male and female BMPR2^R899X mice. Each symbol is a value from one mouse. By multiple ANOVA, considering sex, BMPR2 mutation, and ESR status as factors, BMPR2 mutation increases RVSP (p=0.0003 by MANOVA), ESR1 trends towards restoration (p=0.07), and ESR2 completely restores (p=0.005) RVSP. Sex did not have significant interaction (all mice received 16αOHE1 in pumps). Numbers of mice in each group are listed at the bottom of the plot; mice were age-matched, and controls were done contemporaneously for each group. (C) ESR1 knockout mice partially and ESR2 knockout mice completely reduce muscularized small vessels in BMPR2^R899X mice to normal. By multiple ANOVA, considering sex, BMPR2 mutation, and ESR status as factors, BMPR2 mutation increases muscularized vessels (p=0.0002 by MANOVA), and both ESR1 (p=0.02), and ESR2 (p=0.0005) normalize muscularized vessels. (D) Immunohistochemistry for smooth muscle actin (red) counterstained with DAPI (blue). Apparent intensity of DAPI staining has been reduced to clarify actin. Muscularized and partially muscularized small vessels are roughly doubled in BMPR2^R899X mice, and there is the occasional occluded vessel (see inset). ESR1 knockout reduces muscularization, but still has some occluded vessels (top two in field); ESR2 knockout normalizes muscularization, and occluded vessels are no longer apparent.

Figure 4 –

(A) Anastrozole & Fulvestrant used preventatively result in increased PPARγ (p=0.001) and CD36 (p=0.02) in whole lung, which are otherwise suppressed by R899X mutation (p=0.003 for PPARγ and p=0.007 for CD36) by two way ANOVA. Numbers are densitometry for the bands above; each lane is whole lung from a different animal. (B) Anastrozole & Fulvestrant used as treatment result in increased PPARγ (p=0.019) and CD36 (p=0.027) in whole lung, which are otherwise suppressed by R899X mutation (p=0.047 for PPARγ and p=0.043 for CD36) by two way ANOVA. Numbers are densitometry for the bands above; each lane is whole lung from a different animal. (C) Anastrozole & Fulvestrant (A+F) used as a treatment significantly reduce insulin resistance in R899X mice (p=0.012), while Tamoxifen (Tam) trends towards improvement (p=0.06). Tests are Fisher’s LSD after ANOVA (p=0.01 for rejection of null). Note that insulin resistance in even control mice is quite high, as they are FVB/N mice on western diet. (D) Insulin resistance is highly correlated with right ventricular systolic pressure (correlation=0.71, p<0.0001). Open circles are Rosa26-only controls; grey circles are R899X mice treated with Anastrozole & Fulvestrant; filled circles are R899X mice treated with vehicle.

Figure 5 –

(A) By two way ANOVA, ESR1 knockout does not affect PPARG or CD36 in whole lung, although PPARG is suppressed by R899X mutation (p=.03) (B) By two-way ANOVA, PPARG is suppressed by R899X mutation (p=.02) in whole lung and induced by ESR2 knockout (p=0.001); CD36 is also induced by ESR2 knockout (p=−.001).

Figure 6 –

(A) Insulin mobilization of GLUT4 in mPMVEC is decreased in BMPR2^R899X mice, and decreased further by pretreatment with 16αOHE1. Insulin mobilization was determined by counting perinuclear vs membrane associated GFP-tagged GLUT4 in each of 100 cells in each of three technical replicates (% in each replicate indicated by circles). Every group has three technical replicates: some are overlapping. Error bars are standard deviation; differences are significant at p<0.0001 by ANOVA, with comparisons indicated by Fisher’s LSD. Differences are also significant at p=0.01 by Wilcoxon non-parametric test. (B) Live cell images of mPMVEC transiently transfected with GFP-tagged Glut4, untreated and pretreated with 16αOHE1, either with vehicle or 15 minutes after treatment with insulin (which ought to mobilize Glut4 to the cell surface). (C) Seahorse Extracellular Flux analysis using the Mito Stress Protocol shows an increase in maximum oxygen consumption rate with FCCP treatment (@~40–60 minutes). (D) Quantitation of maximum oxygen consumption rate from data in (C). (E) A Mitosox Red assay suggests that the increased oxygen consumption in Bmpr2 mutant cells with 16αOHE1 is driven by increased mitochondrial superoxide production.