Background-Pulmonary arterial hypertension (PAH) is a hyperproliferative vascular disorder observed predominantly in women. Estrogen is a potent mitogen in human pulmonary artery smooth muscle cells and contributes to PAH in vivo; however, the mechanisms attributed to this causation remain obscure. Curiously, heightened expression of the estrogenmetabolizing enzyme cytochrome P450 1B1 (CYP1B1) is reported in idiopathic PAH and murine models of PAH. Methods and Results-Here, we investigated the putative pathogenic role of CYP1B1 in PAH. Quantitative reverse transcriptionpolymerase chain reaction, immunoblotting, and in situ analysis revealed that pulmonary CYP1B1 is increased in hypoxic PAH, hypoxicϩSU5416 PAH, and human PAH and is highly expressed within the pulmonary vascular wall. PAH was assessed in mice via measurement of right ventricular hypertrophy, pulmonary vascular remodeling, and right ventricular systolic pressure. Hypoxic PAH was attenuated in CYP1B1 Ϫ/Ϫ mice, and the potent CYP1B1 inhibitor 2,3Ј,4,5Ј-tetramethoxystilbene (TMS; 3 mg ⅐ kg Ϫ1 ⅐ d

Rationale: The incidence of pulmonary arterial hypertension is greater in women, suggesting estrogens may play a role in the disease pathogenesis. Experimentally, in males, exogenously administered estrogen can protect against pulmonary hypertension (PH). However, in models that display female susceptibility, estrogens may play a causative role.Objectives: To clarify the influence of endogenous estrogen and sex in PH and assess the therapeutic potential of a clinically available aromatase inhibitor. Methods:We interrogated the effect of reduced endogenous estrogen in males and females using the aromatase inhibitor, anastrozole, in two models of PH: the hypoxic mouse and Sugen 5416/hypoxic rat. We also determined the effects of sex on pulmonary expression of aromatase in these models and in lungs from patients with pulmonary arterial hypertension.Measurements and Main Results: Anastrozole attenuated PH in both models studied, but only in females. To verify this effect was caused by reduced estrogenic activity we confirmed that in hypoxic mice inhibition of estrogen receptor a also has a therapeutic effect specifically in females. Female rodent lung displays increased aromatase and decreased bone morphogenetic protein receptor 2 and Id1 expression compared with male. Anastrozole treatment reversed the impaired bone morphogenetic protein receptor 2 pathway in females. Increased aromatase expression was also detected in female human pulmonary artery smooth muscle cells compared with male.Conclusions: The unique phenotype of female pulmonary arteries facilitates the therapeutic effects of anastrozole in experimental PH confirming a role for endogenous estrogen in the disease pathogenesis in females and suggests aromatase inhibitors may have therapeutic potential.Keywords: pulmonary hypertension; estrogen; sex At a Glance CommentaryScientific Knowledge on the Subject: Females develop pulmonary arterial hypertension (PAH) more frequently than males. The role of estrogen in this female susceptibility is poorly understood.What This Study Adds to the Field: Our research shows that inhibition of endogenous estrogen synthesis using an aromatase inhibitor or inhibition of estrogen receptor a has therapeutic effects and restores bone morphogenetic protein receptor 2 expression in female but not male models of PAH. These findings suggest estrogen plays a pathogenic role in the pathology of PAH specifically in females.

17β oestradiol is critical to the development of PAH and severe hypoxia-induced PAH in female SERT+ mice. In hPASMCs, 17β oestradiol-induced proliferation is dependant on de novo serotonin synthesis and stimulation of the 5-HT(1B) receptor. These interactions between the serotonin system and 17β oestradiol may contribute to the increased risk of PAH associated with female gender.

BackgroundIdiopathic and familial forms of pulmonary arterial hypertension (PAH) occur more frequently in women than men. However, the reason for this remains unknown. Both the calcium binding protein S100A4/Mts1 (Mts1) and its endogenous receptor (receptor for advanced glycosylation end products; RAGE) have been implicated in the development of PAH. We wished to investigate if the Mts1/RAGE pathway may play a role in the gender bias associated with PAH.MethodsWe investigated the effects of gender on development of PAH in mice over-expressing Mts1 (Mts1+ mice) via measurement of pulmonary arterial remodeling, systolic right ventricular pressure (sRVP) and right ventricular hypertrophy (RVH). Gender differences in pulmonary arterial Mts1 and RAGE expression were assessed by qRT-PCR and immunohistochemistry. Western blotting and cell counts were used to investigate interactions between 17β-estradiol, Mts1 and RAGE on proliferation of human pulmonary artery smooth muscle cells (hPASMCs). Statistical analysis was by one-way analysis of variance with Dunnetts post test or two-way analysis of variance with Bonferronis post test, as appropriate.ResultsFemale Mts1+ mice developed increased sRVP and pulmonary vascular remodeling, whereas male Mts1+ mice remained unaffected. The development of plexiform-like lesions in Mts1+ mice was specific to females. These lesions stained positive for both Mts1 and RAGE in the endothelial and adventitial layers. Expression of pulmonary arterial Mts1 was greater in female than male Mts1+ mice, and was localised to the medial and adventitial layers in non plexiform-like pulmonary arteries. RAGE gene expression and immunoreactivity were similar between male and female Mts1+ mice and RAGE staining was localised to the endothelial layer in non plexiform-like pulmonary arteries adjacent to airways. In non-plexiform like pulmonary arteries not associated with airways RAGE staining was present in the medial and adventitial layers. Physiological concentrations of 17β-estradiol increased Mts1 expression in hPASMCs. 17β-estradiol-induced hPASMC proliferation was inhibited by soluble RAGE, which antagonises the membrane bound form of RAGE.ConclusionsMts1 over-expression combined with female gender is permissive to the development of experimental PAH in mice. Up-regulation of Mts1 and subsequent activation of RAGE may contribute to 17β-estradiol-induced proliferation of hPASMCs.