Effect of estrogen receptor α on cardiopulmonary adaptation to chronic developmental hypoxia in a rat model.

Humans living at high-altitude (HA) have adapted to this environment by increasing pulmonary vascular and alveolar growth. RNA sequencing data from a novel murine model that mimics this phenotypical response to HA suggested estrogen signaling via estrogen receptor alpha (ERα) may be involved in this adaptation. We hypothesized ERα was a key mediator in the cardiopulmonary adaptation to chronic hypoxia and sought to delineate the mechanistic role ERα contributes to this process by exposing novel loss-of-function ERα mutant (ERαMut) rats to simulated HA. ERα mutant or wild-type (wt) rats were exposed to normoxia or hypoxia starting at conception and continued postnatally until 6 wk of age. Both wt and ERαMut animals born and raised in hypoxia exhibited lower body mass and higher hematocrits, total alveolar volumes (Va), diffusion capacities of carbon monoxide (DLCO), pulmonary arteriole (PA) wall thickness, and Fulton indices than normoxia animals. Right ventricle adaptation was maintained in the setting of hypoxia. Although no major physiologic differences were seen between wt and ERαMut animals at either exposure, ERαMut animals exhibited smaller mean linear intercepts (MLI) and increased PA total and lumen areas. Hypoxia exposure or ERα loss-of-function did not affect lung mRNA abundance of vascular endothelial growth factor, angiopoietin 2, or apelin. Sexual dimorphisms were noted in PA wall thickness and PA lumen area in ERαMut rats. In summary, in room air-exposed rats and rats with peri- and postnatal hypoxia exposure, ERα loss-of-function was associated with decreased alveolar size (primarily driven by hypoxic animals) and increased PA remodeling.NEW & NOTEWORTHY By exposing novel loss-of-function estrogen receptor alpha (Erα) mutant rats to a novel model of human high-altitude exposure, we demonstrate that ERα has subtle but inconsistent effects on endpoints relevant to cardiopulmonary adaptation to chronic hypoxia. Given that we observed some histologic, sex, and genotype differences, further research into cell-specific effects of ERα during hypoxia-induced cardiopulmonary adaptation is warranted.

Wnt7a deficit is associated with dysfunctional angiogenesis in pulmonary arterial hypertension.

INTRODUCTION: Pulmonary arterial hypertension (PAH) is characterised by loss of microvessels. The Wnt pathways control pulmonary angiogenesis but their role in PAH is incompletely understood. We hypothesised that Wnt activation in pulmonary microvascular endothelial cells (PMVECs) is required for pulmonary angiogenesis, and its loss contributes to PAH. METHODS: Lung tissue and PMVECs from healthy and PAH patients were screened for Wnt production. Global and endothelial-specific Wnt7a -/- mice were generated and exposed to chronic hypoxia and Sugen-hypoxia (SuHx). RESULTS: Healthy PMVECs demonstrated >6-fold Wnt7a expression during angiogenesis that was absent in PAH PMVECs and lungs. Wnt7a expression correlated with the formation of tip cells, a migratory endothelial phenotype critical for angiogenesis. PAH PMVECs demonstrated reduced vascular endothelial growth factor (VEGF)-induced tip cell formation as evidenced by reduced filopodia formation and motility, which was partially rescued by recombinant Wnt7a. We discovered that Wnt7a promotes VEGF signalling by facilitating Y1175 tyrosine phosphorylation in vascular endothelial growth factor receptor 2 (VEGFR2) through receptor tyrosine kinase-like orphan receptor 2 (ROR2), a Wnt-specific receptor. We found that ROR2 knockdown mimics Wnt7a insufficiency and prevents recovery of tip cell formation with Wnt7a stimulation. While there was no difference between wild-type and endothelial-specific Wnt7a -/- mice under either chronic hypoxia or SuHx, global Wnt7a +/- mice in hypoxia demonstrated higher pulmonary pressures and severe right ventricular and lung vascular remodelling. Similar to PAH, Wnt7a +/- PMVECs exhibited an insufficient angiogenic response to VEGF-A that improved with Wnt7a. CONCLUSIONS: Wnt7a promotes VEGF signalling in lung PMVECs and its loss is associated with an insufficient VEGF-A angiogenic response. We propose that Wnt7a deficiency contributes to progressive small vessel loss in PAH.

SOX17 Deficiency Mediates Pulmonary Hypertension: At the Crossroads of Sex, Metabolism, and Genetics.

Rationale: Genetic studies suggest that SOX17 (SRY-related HMG-box 17) deficiency increases pulmonary arterial hypertension (PAH) risk. Objectives: On the basis of pathological roles of estrogen and HIF2α (hypoxia-inducible factor 2α) signaling in pulmonary artery endothelial cells (PAECs), we hypothesized that SOX17 is a target of estrogen signaling that promotes mitochondrial function and attenuates PAH development via HIF2α inhibition. Methods: We used metabolic (Seahorse) and promoter luciferase assays in PAECs together with the chronic hypoxia murine model to test the hypothesis. Measurements and Main Results: Sox17 expression was reduced in PAH tissues (rodent models and from patients). Chronic hypoxic pulmonary hypertension was exacerbated by mice with conditional Tie2-Sox17 (Sox17EC-/-) deletion and attenuated by transgenic Tie2-Sox17 overexpression (Sox17Tg). On the basis of untargeted proteomics, metabolism was the top pathway altered by SOX17 deficiency in PAECs. Mechanistically, we found that HIF2α concentrations were increased in the lungs of Sox17EC-/- and reduced in those from Sox17Tg mice. Increased SOX17 promoted oxidative phosphorylation and mitochondrial function in PAECs, which were partly attenuated by HIF2α overexpression. Rat lungs in males displayed higher Sox17 expression versus females, suggesting repression by estrogen signaling. Supporting 16α-hydroxyestrone (16αOHE; a pathologic estrogen metabolite)-mediated repression of SOX17 promoter activity, Sox17Tg mice attenuated 16αOHE-mediated exacerbations of chronic hypoxic pulmonary hypertension. Finally, in adjusted analyses in patients with PAH, we report novel associations between a SOX17 risk variant, rs10103692, and reduced plasma citrate concentrations (n = 1,326). Conclusions: Cumulatively, SOX17 promotes mitochondrial bioenergetics and attenuates PAH, in part, via inhibition of HIF2α. 16αOHE mediates PAH development via downregulation of SOX17, linking sexual dimorphism and SOX17 genetics in PAH.

Sex differences in right ventricular adaptation to pressure overload in a rat model.

With severe right ventricular (RV) pressure overload, women demonstrate better clinical outcomes compared with men. The mechanoenergetic mechanisms underlying this protective effect, and their dependence on female endogenous sex hormones, remain unknown. To investigate these mechanisms and their impact on RV systolic and diastolic functional adaptation, we created comparable pressure overload via pulmonary artery banding (PAB) in intact male and female Wistar rats and ovariectomized (OVX) female rats. At 8 wk after surgery, right heart catheterization demonstrated increased RV energy input [indexed pressure-volume area (iPVA)] in all PAB groups, with the greatest increase in intact females. PAB also increased RV energy output [indexed stroke or external work (iEW)] in all groups, again with the greatest increase in intact females. In contrast, PAB only increased RV contractility-indexed end-systolic elastance (iEes)] in females. Despite these sex-dependent differences, no statistically significant effects were observed in the ratio of RV energy output to input (mechanical efficiency) or in mechanoenergetic cost to pump blood with pressure overload. These metrics were similarly unaffected by loss of endogenous sex hormones in females. Also, despite sex-dependent differences in collagen content and organization with pressure overload, decreases in RV compliance and relaxation time constant (tau Weiss) were not determined to be sex dependent. Overall, despite sex-dependent differences in RV contractile and fibrotic responses, RV mechanoenergetics for this degree and duration of pressure overload are comparable between sexes and suggest a homeostatic target.NEW & NOTEWORTHY Sex differences in right ventricular mechanical efficiency and energetic adaptation to increased right ventricular afterload were measured. Despite sex-dependent differences in contractile and fibrotic responses, right ventricular mechanoenergetic adaptation was comparable between the sexes, suggesting a homeostatic target.

Association of premature menopause with incident pulmonary hypertension: A cohort study.

BACKGROUND: Several forms of pulmonary hypertension (PH) disproportionately affect women. Animal and human studies suggest that estradiol exerts mixed effects on the pulmonary vasculature. Whether premature menopause represents a risk factor for PH is unknown. METHODS AND FINDINGS: In this cohort study, women in the UK Biobank aged 40-69 years who were postmenopausal and had complete data available on reproductive history were included. Premature menopause, defined as menopause occurring before age 40 years. Postmenopausal women without premature menopause served as the reference group. The primary outcome was incident PH, ascertained by appearance of a qualifying ICD code in the participant's UK Biobank study record. Of 136,715 postmenopausal women included, 5,201 (3.8%) had premature menopause. Participants were followed up for a median of 11.1 (interquartile range 10.5-11.8) years. The primary outcome occurred in 38 women (0.73%) with premature menopause and 409 (0.31%) without. After adjustment for age, race, ever-smoking, body-mass index, systolic blood pressure, antihypertensive medication use, non-high-density lipoprotein cholesterol, cholesterol-lowering medication use, C-reactive protein, prevalent type 2 diabetes, obstructive sleep apnea, heart failure, mitral regurgitation, aortic stenosis, venous thromboembolism, forced vital capacity (FVC), the forced expiratory volume in 1 second-to-FVC ratio, use of menopausal hormone therapy, and hysterectomy status, premature menopause was independently associated with PH (hazard ratio 2.13, 95% CI 1.31-3.23, P<0.001). In analyses of alternate menopausal age thresholds, risk of PH appeared to increase progressively with younger age at menopause (Ptrend <0.001), with 4.8-fold risk in women with menopause before age 30 years (95% CI 1.82-12.74, P = 0.002). Use of menopausal hormone therapy did not modify the association of premature menopause with PH. CONCLUSIONS: Premature menopause may represent an independent risk factor for PH in women. Further investigation of the role of sex hormones in PH is needed in animal and human studies to elucidate pathobiology and identify novel therapeutic targets.

17ß-Estradiol and estrogen receptor α protect right ventricular function in pulmonary hypertension via BMPR2 and apelin.

Women with pulmonary arterial hypertension (PAH) exhibit better right ventricular (RV) function and survival than men; however, the underlying mechanisms are unknown. We hypothesized that 17ß-estradiol (E2), through estrogen receptor α (ER-α), attenuates PAH-induced RV failure (RVF) by upregulating the procontractile and prosurvival peptide apelin via a BMPR2-dependent mechanism. We found that ER-α and apelin expression were decreased in RV homogenates from patients with RVF and from rats with maladaptive (but not adaptive) RV remodeling. RV cardiomyocyte apelin abundance increased in vivo or in vitro after treatment with E2 or ER-α agonist. Studies employing ER-α-null or ER-ß-null mice, ER-α loss-of-function mutant rats, or siRNA demonstrated that ER-α is necessary for E2 to upregulate RV apelin. E2 and ER-α increased BMPR2 in pulmonary hypertension RVs and in isolated RV cardiomyocytes, associated with ER-α binding to the Bmpr2 promoter. BMPR2 is required for E2-mediated increases in apelin abundance, and both BMPR2 and apelin are necessary for E2 to exert RV-protective effects. E2 or ER-α agonist rescued monocrotaline pulmonary hypertension and restored RV apelin and BMPR2. We identified what we believe to be a novel cardioprotective E2/ER-α/BMPR2/apelin axis in the RV. Harnessing this axis may lead to novel RV-targeted therapies for PAH patients of either sex.

Releasing the brakes: a case report of pulmonary arterial hypertension induced by immune checkpoint inhibitor therapy.

Immune checkpoint inhibitors successfully treat various malignancies by inducing an immune response to tumor cells. However, their use has been associated with a variety of autoimmune disorders, such as diabetes, hepatitis, and pneumonitis. Pulmonary arterial hypertension due to checkpoint inhibitor use has not yet been described. We present a novel case of pulmonary arterial hypertension associated with systemic lupus erythematosus and Sjogren's syndrome overlap that was induced by therapy with the checkpoint inhibitor durvalumab.

At the X-Roads of Sex and Genetics in Pulmonary Arterial Hypertension.

Group 1 pulmonary hypertension (pulmonary arterial hypertension; PAH) is a rare disease characterized by remodeling of the small pulmonary arteries leading to progressive elevation of pulmonary vascular resistance, ultimately leading to right ventricular failure and death. Deleterious mutations in the serine-threonine receptor bone morphogenetic protein receptor 2 (BMPR2; a central mediator of bone morphogenetic protein (BMP) signaling) and female sex are known risk factors for the development of PAH in humans. In this narrative review, we explore the complex interplay between the BMP and estrogen signaling pathways, and the potentially synergistic mechanisms by which these signaling cascades increase the risk of developing PAH. A comprehensive understanding of these tangled pathways may reveal therapeutic targets to prevent or slow the progression of PAH.

Estrogen receptor-α prevents right ventricular diastolic dysfunction and fibrosis in female rats.

Although women are more susceptible to pulmonary arterial hypertension (PAH) than men, their right ventricular (RV) function is better preserved. Estrogen receptor-α (ERα) has been identified as a likely mediator for estrogen protection in the RV. However, the role of ERα in preserving RV function and remodeling during pressure overload remains poorly understood. We hypothesized that loss of functional ERα removes female protection from adverse remodeling and is permissive for the development of a maladapted RV phenotype. Male and female rats with a loss-of-function mutation in ERα (ERαMut) and wild-type (WT) littermates underwent RV pressure overload by pulmonary artery banding (PAB). At 10 wk post-PAB, WT and ERαMut demonstrated RV hypertrophy. Analysis of RV pressure waveforms demonstrated RV-pulmonary vascular uncoupling and diastolic dysfunction in female, but not male, ERαMut PAB rats. Similarly, female, but not male, ERαMut exhibited increased RV fibrosis, comprised primarily of thick collagen fibers. There was an increased protein expression ratio of TIMP metallopeptidase inhibitor 1 (Timp1) to matrix metalloproteinase 9 (Mmp9) in female ERαMut compared with WT PAB rats, suggesting less collagen degradation. RNA-sequencing in female WT and ERαMut RV revealed kallikrein-related peptidase 10 (Klk10) and Jun Proto-Oncogene (Jun) as possible mediators of female RV protection during PAB. In summary, ERα in females is protective against RV-pulmonary vascular uncoupling, diastolic dysfunction, and fibrosis in response to pressure overload. ERα appears to be dispensable for RV adaptation in males. ERα may be a mediator of superior RV adaptation in female patients with PAH.NEW & NOTEWORTHY Using a novel loss-of-function mutation in estrogen receptor-α (ERα), we demonstrate that female, but not male, ERα mutant rats display right ventricular (RV)-vascular uncoupling, diastolic dysfunction, and fibrosis following pressure overload, indicating a sex-dependent role of ERα in protecting against adverse RV remodeling. TIMP metallopeptidase inhibitor 1 (Timp1), matrix metalloproteinase 9 (Mmp9), kallikrein-related peptidase 10 (Klk10), and Jun Proto-Oncogene (Jun) were identified as potential mediators in ERα-regulated pathways in RV pressure overload.

Sex, Gender, and Sex Hormones in Pulmonary Hypertension and Right Ventricular Failure.

Pulmonary hypertension (PH) encompasses a syndrome of diseases that are characterized by elevated pulmonary artery pressure and pulmonary vascular remodeling and that frequently lead to right ventricular (RV) failure and death. Several types of PH exhibit sexually dimorphic features in disease penetrance, presentation, and progression. Most sexually dimorphic features in PH have been described in pulmonary arterial hypertension (PAH), a devastating and progressive pulmonary vasculopathy with a 3-year survival rate <60%. While patient registries show that women are more susceptible to development of PAH, female PAH patients display better RV function and increased survival compared to their male counterparts, a phenomenon referred to as the "estrogen paradox" or "estrogen puzzle" of PAH. Recent advances in the field have demonstrated that multiple sex hormones, receptors, and metabolites play a role in the estrogen puzzle and that the effects of hormone signaling may be time and compartment specific. While the underlying physiological mechanisms are complex, unraveling the estrogen puzzle may reveal novel therapeutic strategies to treat and reverse the effects of PAH/PH. In this article, we (i) review PH classification and pathophysiology; (ii) discuss sex/gender differences observed in patients and animal models; (iii) review sex hormone synthesis and metabolism; (iv) review in detail the scientific literature of sex hormone signaling in PAH/PH, particularly estrogen-, testosterone-, progesterone-, and dehydroepiandrosterone (DHEA)-mediated effects in the pulmonary vasculature and RV; (v) discuss hormone-independent variables contributing to sexually dimorphic disease presentation; and (vi) identify knowledge gaps and pathways forward. © 2020 American Physiological Society. Compr Physiol 10:125-170, 2020.

A case report of hepatopulmonary syndrome in hereditary hemorrhagic telangiectasia (HHT): Not all right-to-left shunting in HHT is due to pulmonary arteriovenous malformations.

RATIONALE: Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disorder characterized by abnormal vessel growth that results in telangiectasias and arteriovenous malformations (AVMs) in the skin, mucosa, and viscera. Up to 30% of patients with HHT exhibit pulmonary AVMs (PAVMs), clinically manifesting as right-to-left shunting and hypoxemia. PATIENT CONCERNS: We report an unusual and novel case of a patient with HHT who lacked clinical sequelae of portal hypertension but presented to clinic with hypoxemia without dyspnea. DIAGNOSES: Diagnostic workup revealed noncardiac right-to-left shunting due to hepatopulmonary syndrome (HPS) from HHT-induced portal hypertension rather than PAVMs. The diagnosis was confirmed by the absence of PAVMs on chest computed tomography and evidence of elevated portal pressures as noted by the presence of small esophageal varices on upper endoscopy and histologic findings on liver biopsy. INTERVENTION: Due to the patient's mild symptoms, no further intervention was required. He was closely followed up in the outpatient setting for changes in symptoms and underwent annual screening for development of PAVMs. OUTCOMES: The patient continues to do well clinically. He has not experienced worsening hypoxemia or dyspnea and has not developed PAVMs. LESSONS: Given that management of hypoxemia in HPS drastically differs from that of hypoxemia due to PAVMs, this case demonstrates the importance of evaluating HHT patients for HPS if they exhibit impaired oxygenation and noncardiac right-to-leftshunting in the setting of hepatic arteriovenous shunting.

Female Sex and Gender in Lung/Sleep Health and Disease. Increased Understanding of Basic Biological, Pathophysiological, and Behavioral Mechanisms Leading to Better Health for Female Patients with Lung Disease.

Female sex/gender is an undercharacterized variable in studies related to lung development and disease. Notwithstanding, many aspects of lung and sleep biology and pathobiology are impacted by female sex and female reproductive transitions. These may manifest as differential gene expression or peculiar organ development. Some conditions are more prevalent in women, such as asthma and insomnia, or, in the case of lymphangioleiomyomatosis, are seen almost exclusively in women. In other diseases, presentation differs, such as the higher frequency of exacerbations experienced by women with chronic obstructive pulmonary disease or greater cardiac morbidity among women with sleep-disordered breathing. Recent advances in -omics and behavioral science provide an opportunity to specifically address sex-based differences and explore research needs and opportunities that will elucidate biochemical pathways, thus enabling more targeted/personalized therapies. To explore the status of and opportunities for research in this area, the NHLBI, in partnership with the NIH Office of Research on Women's Health and the Office of Rare Diseases Research, convened a workshop of investigators in Bethesda, Maryland on September 18 and 19, 2017. At the workshop, the participants reviewed the current understanding of the biological, behavioral, and clinical implications of female sex and gender on lung and sleep health and disease, and formulated recommendations that address research gaps, with a view to achieving better health outcomes through more precise management of female patients with nonneoplastic lung disease. This report summarizes those discussions.

Hypoxia Upregulates Estrogen Receptor ß in Pulmonary Artery Endothelial Cells in a HIF-1α-Dependent Manner.

17ß-Estradiol (E2) attenuates hypoxia-induced pulmonary hypertension (HPH) through estrogen receptor (ER)-dependent effects, including inhibition of hypoxia-induced endothelial cell proliferation; however, the mechanisms responsible for this remain unknown. We hypothesized that the protective effects of E2 in HPH are mediated through hypoxia-inducible factor 1α (HIF-1α)-dependent increases in ERß expression. Sprague-Dawley rats and ERα or ERß knockout mice were exposed to hypobaric hypoxia for 2-3 weeks. The effects of hypoxia were also studied in primary rat or human pulmonary artery endothelial cells (PAECs). Hypoxia increased expression of ERß, but not ERα, in lungs from HPH rats as well as in rat and human PAECs. ERß mRNA time dependently increased in PAECs exposed to hypoxia. Normoxic HIF-1α/HIF-2α stabilization increased PAEC ERß, whereas HIF-1α knockdown decreased ERß abundance in hypoxic PAECs. In turn, ERß knockdown in hypoxic PAECs increased HIF-2α expression, suggesting a hypoxia-sensitive feedback mechanism. ERß knockdown in hypoxic PAECs also decreased expression of the HIF inhibitor prolyl hydroxylase 2 (PHD2), whereas ERß activation increased PHD2 and decreased both HIF-1α and HIF-2α, suggesting that ERß regulates the PHD2/HIF-1α/HIF-2α axis during hypoxia. Whereas hypoxic wild-type or ERα knockout mice treated with E2 demonstrated less pulmonary vascular remodeling and decreased HIF-1α after hypoxia compared with untreated hypoxic mice, ERß knockout mice exhibited increased HIF-2α and an attenuated response to E2 during hypoxia. Taken together, our results demonstrate a novel and potentially therapeutically targetable mechanism whereby hypoxia, via HIF-1α, increases ERß expression and the E2-ERß axis targets PHD2, HIF-1α, and HIF-2α to attenuate HPH development.

Isolated heart model demonstrates evidence of contractile and diastolic dysfunction in right ventricles from rats with sugen/hypoxia-induced pulmonary hypertension.

Although extensively used for the study of left ventricular function, limited experience exists with the isolated heart model in the evaluation of right ventricular (RV) function. In particular, no published experience exists with this tool in sugen/hypoxia-induced pulmonary hypertension (SuHx-PH), a frequently used model of severe and progressive PH We sought to characterize markers of RV contractile and diastolic function in SuHx-PH and to establish their relationship with markers of maladaptive RV remodeling. Hearts were excised from anesthetized Sprague Dawley rats with or without SuHx-PH and perfused via the aorta using a Langendorff preparation. We explored the Frank-Starling relationship of RV function (RV developed pressure, dP/dtmax, and dP/dtmin; all normalized to RV mass) by increasing RV end-diastolic pressure (RVEDP) from 0 to 40 mmHg. Functional studies were complemented by quantification of RV pro-apoptotic signaling (bcl2/bax), procontractile signaling (apelin), and stress response signaling (p38MAPK activation). Pearson's correlation analysis was performed for functional and biochemical parameters. SuHx-RVs exhibited severe RV dysfunction with marked hypertrophy and decreased echocardiographic cardiac output. For any given RVEDP, SuHx-RVs demonstrated less developed pressure and lower dP/dtmax, as well as less pronounced dP/dtmin, suggestive of decreased contractile and diastolic function. SuHx-RVs exhibited decreased bcl2/bax ratios, apelin expression, and p38MAPK activation. Bcl2/bax and apelin RNA abundance correlated positively with RV developed pressure and dP/dtmax and negatively with dP/dtmin p38MAPK activation correlated positively with RV developed pressure. We conclude that SuHx-RVs exhibit severe contractile and diastolic dysfunction. Increased pro-apoptotic signaling and attenuated procontractile and stress response signaling may contribute to these functional alterations.