Female Pulmonary Arterial Hypertension Research Articles

Dynamically stiffening biomaterials reveal age- and sex-specific differences in pulmonary arterial adventitial fibroblast activation

Respiratory diseases like pulmonary arterial hypertension (PAH) frequently exhibit sexual dimorphism. Female PAH patients are more susceptible to the disease but have increased survival rates. This phenomenon is known as the estrogen paradox, and the underlying mechanisms are not fully understood. During PAH progression in vivo, human pulmonary arterial adventitial fibroblasts (hPAAFs) differentiate into an activated phenotype. These cells produce excess, aberrant extracellular matrix proteins that stiffen the surrounding pulmonary arterial tissues. Here, we employed dynamic poly(ethylene glycol)-alpha methacrylate (PEGαMA)-based biomaterials to study how the age and sex of human serum influenced hPAAF activation in response to microenvironmental stiffening in vitro. Results showed female and male cells responded differently to increases in microenvironmental stiffness and serum composition. Male hPAAFs were less activated than female cells on soft hydrogels and more responsive to increases in microenvironmental stiffness regardless of serum composition. Female hPAAF activation followed this pattern only when cultured in younger (age < 50) female serum or when older (age ≥ 50) female serum was supplemented with estradiol. Otherwise, female hPAAF activation was relatively high on both soft and stiffened hydrogels, with little difference in activation between the two conditions. Collectively, these results suggest that it may be possible to model the estrogen paradox observed in PAH in vitro and that it is critical for researchers to report cell sex and serum source when conducting in vitro experimentation.

Physiological and Psychological Response to Acute Mental Stress in Female Patients Affected by Chronic Pulmonary Arterial Hypertension: An Explorative Controlled Pilot Trial.

Little is known about physiological and psychological responses to mental stress in stable patients affected by pulmonary arterial hypertension (PAH). The current explorative controlled pilot study was conducted to investigate whether heart rate (HR) and perceived stress would differ during standardized mental stress testing in PAH patients compared to healthy subjects. Correlation analysis between HR, perceived stress, participants' psychological status and performance on the mental stress task was also performed. The study included 13 female PAH patients (average age: 44.38 ± 10.88 years; average education: 14 ± 3.07 years; mean duration of illness: 9.15 ± 5.37 years) and 13 female controls similar in age (mean age: 47.85 ± 6.36 years) and education (15.92 ± 1.55 years). Participants performed a standardized 9 min mental stress test (computer based, adaptive math task). HR and perceived stress during the task were compared to resting baseline and correlated with psychological state and task performance. Both HR and perceived stress significantly increased during mental stress in a similar way in both groups. A significant correlation was found between HR and perceived stress. Our data show that moderate mental stress has a comparable effect on HR and perceived stress increase in stable PAH patients and control subjects.

Abstract 13266: Sex Specific Differences in the Gut Microbiome in Patients With Pulmonary Arterial Hypertension

Introduction: Pulmonary arterial hypertension (PAH) patients exhibit sexual dimorphism in pulmonary vascular remodeling and right ventricular (RV) dysfunction. While female PAH patients have severe pulmonary vascular remodeling, male PAH patients have worse RV function. Inflammation, a key component of PAH pathobiology, has been implicated as a potential mechanism for sexual dimorphism. Gut dysbiosis causes systemic inflammation. However, it is unknown whether gut dysbiosis contributes to sexual dimorphism in PAH. Hypothesis: There are sex-specific differences in the gut microbiome and microbial metabolites of PAH patients. Methods: 16S ribosomal ribonucleic acid gene sequencing was performed on stool samples from PAH patients (n=73). Markers of inflammation and circulating microbial metabolites were measured using plasma samples. Results: There was no significant difference in the Shannon diversity indices or species richness (Chao1 indices) between the gut microbiomes of male and female PAH patients. Moreover, principal component analysis of pairwise Bray-Curtis dissimilarity indices revealed no distinct microbiome compositions dependent on sex. There was no difference in serum claudin-3, a marker of gut permeability, and serum interleukin 6 levels between male and female PAH patients. When examining microbial metabolites, including trimethylamine N-oxide, short chain fatty acids, and secondary bile acids, no sex-specific differences were observed. Linear discriminant analysis of effect size revealed that there were no taxonomic differences based on sex. Conclusions: Male and female PAH patients do not possess distinct differences in their gut microbiomes or metabolites. There are also no sex-specific taxonomic differences. These findings do not support our hypothesis that gut dysbiosis may serve as a possible explanation for the sexual dimorphism observed in PAH.

Relationships between sexual function, mental health, and quality of life of female patients with pulmonary arterial hypertension associated with congenital heart disease.

In recent years, the treatment of pulmonary arterial hypertension (PAH) has gradually increased, including new drugs and surgical methods, the mortality rate of PAH patients has significantly decreased, and the average survival rate has significantly improved. However, there was no obvious improvement in sexual health, mental health, and quality of life (QoL) in patients with PAH.Although an important dimension of QoL, little is known about sexual health and sexual health-related QoLof patients with PAH in China. In this study, the female sexual function index (FSFI)scale, the Symptom Checklist-90 (SCL-90), and emPHasis-10 were used to evaluate PAH associated with congenital heart disease (CHD-PAH) patients' sexual function, mental health, and QoL. The score of sexual function in female CHD-PAH patients ranged from 4.40 to 34.80 points, and the average score was 26.80 (19.00-27.80) points. The detection rate of sexual dysfunction was 48.30%. The FSFIscore of all dimensions of the sexual dysfunction group was significantly lower than that of the nonsexual dysfunction group. In addition, the scores of SCL-90 and emPHasis-10 were significantly higher than that of the nonsexual dysfunction group (p < 0.01). The sexual function was negatively correlated with mental health (r = -0.58,p < 0.01) and QoL (r = -0.62,p < 0.01) in female CHD-PAH patients. The sexual function of female patients with CHD-PAH is not optimistic. Sexual health may impact mental health and overall QoL in female PAH patients. Reasonable intervention measures should be taken to improve their sexual health, so as to improve their overall QoL.

Y-Chromosome Gene, Uty, Protects Against Pulmonary Hypertension by Reducing Proinflammatory Chemokines.

Rationale: Idiopathic pulmonary arterial hypertension (PAH) is a terminal pulmonary vascular disease characterized by increased pressure, right ventricular failure, and death. PAH exhibits a striking sex bias and is up to four times more prevalent in females. Understanding the molecular basis behind sex differences could help uncover novel therapies. Objectives: We previously discovered that the Y chromosome is protective against hypoxia-induced experimental pulmonary hypertension (PH), which may contribute to sex differences in PAH. Here, we identify the gene responsible for Y-chromosome protection, investigate key downstream autosomal genes, and demonstrate a novel preclinical therapy. Methods: To test the effect of Y-chromosome genes on PH development, we knocked down each Y-chromosome gene expressed in the lung by means of intratracheal instillation of siRNA in gonadectomized male mice exposed to hypoxia and monitored changes in right ventricular and pulmonary artery hemodynamics. We compared the lung transcriptome of Uty knockdown mouse lungs to those of male and female PAH patient lungs to identify common downstream pathogenic chemokines and tested the effects of these chemokines on human pulmonary artery endothelial cells. We further inhibited the activity of these chemokines in two preclinical pulmonary hypertension models to test the therapeutic efficacy. Measurements and Main Results: Knockdown of the Y-chromosome gene Uty resulted in more severe PH measured by increased right ventricular pressure and decreased pulmonary artery acceleration time. RNA sequencing revealed an increase in proinflammatory chemokines Cxcl9 and Cxcl10 as a result of Uty knockdown. We found CXCL9 and CXCL10 significantly upregulated in human PAH lungs, with more robust upregulation in females with PAH. Treatment of human pulmonary artery endothelial cells with CXCL9 and CXCL10 triggered apoptosis. Inhibition of Cxcl9 and Cxcl10 expression in male Uty knockout mice and CXCL9 and CXCL10 activity in female rats significantly reduced PH severity. Conclusions:Uty is protective against PH. Reduction of Uty expression results in increased expression of proinflammatory chemokines Cxcl9 and Cxcl10, which trigger endothelial cell death and PH. Inhibition of CLXC9 and CXLC10 rescues PH development in multiple experimental models.

Up-Regulation of the Long Noncoding RNA X-Inactive–Specific Transcript and the Sex Bias in Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a sex-biased disease. Increased expression and activity of the long-noncoding RNA X-inactive-specific transcript (Xist), essential for X-chromosome inactivation and dosage compensation of X-linked genes, may explain the sex bias of PAH. The present studies used a murine model of plexiform PAH, the intersectin-1s (ITSN) heterozygous knockout (KOITSN+/-) mouse transduced with an ITSN fragment (EHITSN) possessing endothelial cell proliferative activity, in conjunction with molecular, cell biology, biochemical, morphologic, and functional approaches. The data demonstrate significant sex-centered differences with regard to EHITSN-induced alterations in pulmonary artery remodeling, lung hemodynamics, and p38/ETS domain containing protein/c-Fos signaling, altogether leading to a more severe female lung PAH phenotype. Moreover, the long-noncoding RNA-Xist is up-regulated in the lungs of female EHITSN-KOITSN+/- mice compared with that in female wild-type mice, leading to sex-specific modulation of the X-linked gene ETS domain containing protein and its target, two molecular events also characteristic to female human PAH lung. More importantly, cyclin A1 expression in the S and G2/M phases of the cell cycle of synchronized pulmonary artery endothelial cells of female PAH patients is greater versus controls, suggesting functional hyperproliferation. Thus, Xist up-regulation leading to female pulmonary artery endothelial cell sexual dimorphic behavior may provide a better understanding of the origin of sex bias in PAH. Notably, the EHITSN-KOITSN+/- mouse is a unique experimental animal model of PAH that recapitulates most of the sexually dimorphic characteristics of human disease.

Sex-dependent right ventricular hypertrophic gene changes after methamphetamine treatment in mice

Methamphetamine (MA) abuse is associated with the development of pulmonary arterial hypertension (PAH) and subsequent right ventricular failure. A recent clinical study demonstrated that female sex is a major risk factor for MA-induced PAH. The mechanisms associated with increased prevalence and severity of MA-induced PAH in females are still unclear. We hypothesized that MA may promote changes in gene expression in the right ventricle contributing to the development and/or worsening of PAH in females. Male and female C57BL/6 mice were treated with either MA or vehicle. Right and left ventricular systolic pressures (RVSP and LVSP, respectively) were assessed and tissue samples were collected for gene expression and histology. LVSP and RVSP were not affected by MA in either males or females. Right ventricular hypertrophy was significantly increased by MA in females but it was not affected by MA in males. In the female mice, MA-induced right ventricular hypertrophy was associated with increased expression of brain natriuretic peptide gene and members of the TGF-β receptor signaling pathway such as TGF-β receptor-1, smad3 and smad7. In male mice, there were no changes in right ventricular gene expression. Our results suggest that MA caused right ventricular hypertrophy in female mice, but not in males and that this was associated with an increase in hypertrophic genes. The right ventricular hypertrophy was not dependent on increased RVSP suggesting a direct effect of MA on the right ventricle. If this translates to PAH patients, it might explain the poor outcome observed in MA-associated female PAH patients.

Abstract MP172: Y-chromosome Gene, Uty, Confers Male Protection Against Pulmonary Hypertension by Mediating Pro-inflammatory Chemokine Effects

Background: Idiopathic pulmonary arterial hypertension (PAH) is a terminal vascular lung disease characterized by increased PA pressure resulting in right ventricular (RV) failure and death. For reasons largely unknown, PAH is up to 4x more prevalent in females. We published that the Y-Chromosome (Chrm) is protective against hypoxia (Hx)-induced pulmonary hypertension (PH) in mice and identified four Y-Chrm genes expressed in the lung. Here, we identify the protective Y-Chrm gene, investigate its mechanism and demonstrate a novel therapeutic approach. Methods and Results: To test the effect of Y-Chrm candidate genes on PH development, we knocked down (KD) each Y-Chrm gene via sequential intratracheal instillation of siRNA in the lungs of male mice exposed to Hx. KD of Y-Chrm gene Uty, but none of the other genes, resulted in more severe PH measured by increased RV systolic pressure (RVSP) and decreased pulmonary arterial acceleration time (PAAT) compared to control (n=4/group; RVSP: Uty-KD= 48.93mmHg, Control= 37.33mmHg, p=0.04; PAAT: Uty-KD= 11.88ms, Control= 14.43ms, p=0.01). RNAseq analysis (Uty-KD vs Control) revealed an increase in pro-inflammatory cytokines Cxcl9 (Log2FC: 1.3, p-adjusted=0.05) and Cxcl10 (Log2FC: 0.9, p-adjusted=0.002). We found Cxcl9/10 significantly upregulated in human PAH lungs vs healthy and female PAH lungs vs male (RT-qPCR). Fluorescent in-situ hybridization revealed Uty and Cxcl9/10 expression co-localized in CD68 + macrophages within the lung. Treatment of human pulmonary artery endothelial cells (PAEC) with Cxcl9/10 resulted in significantly increased apoptosis. We inhibited Cxcl9/10 activity in female rats 14 days post monocrotaline (MCT) injection by treating them with a small molecular inhibitor. Treated rats (n=6) had less severe PH than vehicle-treated controls (n=5) when measured 28-days post MCT (RVSP: Treated= 36.75mmHg, Vehicle=43.62 mmHg, p=0.03; PAAT: Treated= 26.7ms, Vehicle=20 ms, p=0.01; RV hypertrophy index (RV/(LV+IVS)): Treated= 0.48, Vehicle= 0.35, p=0.01). Conclusion: Y-Chrm gene, Uty, is protective against PH. Uty inhibition increases pro-inflammatory cytokines Cxcl9/10 which contribute to PAEC death. Inhibition of Cxcl9/10 activity may provide a novel therapy for the treatment of PH.

Y‐Chromosome Gene, Uty, Protects Against Pulmonary Hypertension by Reducing Lung Pro‐Inflammatory Cytokines

RATIONALEIdiopathic pulmonary arterial hypertension (PAH) is a terminal vascular lung disease up to 4x more common in females. We published that the Y‐Chromosome (Chrm) is protective against hypoxia (Hx)‐induced pulmonary hypertension (PH) in mice and identified four Y‐Chrm genes expressed in the lung. Here, we identify Uty as the protective Y‐Chrm gene and investigate its mechanism of protection against PH to illuminate novel therapeutic targets.METHODS AND RESULTSTo test the effect of Y‐Chrm candidate genes on PH development, each of the four candidate YChrm genes were independently knocked‐down in the lungs of male mice exposed to Hxinduced PH via sequential intratracheal instillation of siRNA. Knockdown of Y‐Chrm gene Uty, but none of the other genes, resulted in more severe PH measured by increased right ventricular systolic pressure (RVSP) and decreased pulmonary arterial acceleration time (PAAT) when compared to experimental controls (n=4/group; RVSP: Uty‐KD= 48.93 mmHg, Control= 37.33 mmHg, p=0.04; PAAT: Uty‐KD= 11.88 ms, Control= 14.43 ms, p= 0.01). Pathway enrichment analysis of RNAseq data (Uty‐Knockdown vs Control) revealed an induction of proinflammatory cytokines including Cxcl9 (Log2FC: 1.3, p‐adjusted= 0.05) and Cxcl10 (Log2FC: 0.9, p‐adjusted=0.002). We found Cxcl9 and Cxcl10 were also significantly up‐regulated in human PAH lungs vs healthy (RT‐qPCR) and female PAH lungs vs male (GSE15197). Fluorescent in‐situ hybridization revealed Uty expression co‐localized with CD68+ macrophages, Cxcl9 and Cxcl10 in the lung. Upon stimulation, macrophages derived from bone marrow of male Uty‐knockout mice displayed increased expression of Cxcl9 and Cxcl10 compared to wildtype mice. Human pulmonary artery endothelial cells treated with Cxcl9 and Cxcl10 in‐vitro exhibited significantly increased cell death compared to those treated with vehicle.CONCLUSIONY‐Chrm gene, Uty, is protective against PH development and may explain Y‐Chrm protection in PH. Since Uty inhibition (both in‐vitro and in‐vivo) results in an increase in pro‐inflammatory cytokines, we hypothesize that a reduction or absence of Uty in the lungs of PAH patients or females results in an increase in lung pro‐inflammatory cytokines which contributes to vascular endothelial dysfunction and more severe PH.Support or Funding InformationAmerican Heart Association grant 17PRE33420159 (C.M.C.), Iris Cantor‐UCLA Women’s Health Center Executive Advisory Board NCATS UCLA CTSI Grant Number UL1TR001881 (C.M.C.), and NIH grants R01HL129051 (M.E.), R01HL131182 (M.E. and A.P.A.).

Potential mechanisms and serum biomarkers involved in sex differences in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a disease associated with high mortality, but notable sex differences have been observed between males and females. For this reason, further research on the mechanisms underlying sex differences in PAH is required to better understand and treat the disease. This study mainly focused on gene expression levels to investigate potential differences in the pathogenesis and progression of PAH between the male and female sexes.Sex-specific differentially expressed genes (DEGs) were analyzed using the Gene Expression Omnibus datasets GSE117261 and GSE38267. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted, and a protein-protein interaction (PPI) network was established based on the identified DEGs to predict potential mechanisms involved in the observed sex differences in the pathogenesis of PAH.We identified 26 female- and 53 male-specific DEGs from lung tissue and 498 female-specific DEGs in blood samples. No male-specific DEGs were identified from blood samples. GO and KEGG pathway analyses revealed that female-specific DEGs in lung tissue were enriched in inflammatory response and cytokine-cytokine receptor interaction, whereas male-specific DEGs were mainly enriched in cellular chemotaxis and the nuclear factor of kappa light polypeptide gene enhancer in B-cell (NF-kappa B) signaling pathway. Lipocalin 2 (LCN2) was the only gene that was differentially expressed in both the lung tissue and the blood of female patients.In conclusion, inflammation and immunity may play key roles in the pathogenesis of female PAH, and LCN2 may act as a serum biomarker of female PAH, whereas the pathogenesis in males is more complicated.

Differential effects of integrin-linked kinase inhibitor Cpd22 on severe pulmonary hypertension in male and female rats.

Pulmonary arterial hypertension (PAH) is a progressive fatal disease with no cure. Inhibition of integrin-linked kinase (ILK) reverses experimental pulmonary hypertension (PH) in male mice, but its effect on severe experimental PH in either male or female animals is unknown. We examined effects of ILK inhibitor Cpd22 on rats with SU5416/hypoxia-induced PH; treatment was performed at six to eight weeks after PH initiation. Five weeks after PH initiation, male and female rats developed similar levels of PH. Eight weeks after PH induction, vehicle-treated male rats had more severe PH than females. Cpd22-treated males, but not females, showed complete suppression of phospho-Akt in small pulmonary arteries (PAs), significantly lower PA medial thickness and percentage of fully occluded arteries, decreased systolic right ventricle (RV) pressure, PA pressure, RV hypertrophy, RV end-diastolic pressure, and improved RV contractility index compared to vehicle-treated group. Cpd22 suppressed proliferation of human male and female PAH pulmonary artery vascular smooth muscle cell (PAVSMC). 17β-estradiol had no effect as a single agent but significantly attenuated Cpd22-dependent inhibition of proliferation in female, but not male, PAH PAVSMC. Taken together, these data demonstrate that male rats develop more severe PH than females but respond better to Cpd22 treatment by reducing pulmonary vascular remodeling, PH, and RV hypertrophy and improving RV functional outcomes. 17β-estradiol diminishes anti-proliferative effect of Cpd22 in female, but not male, human PAH PAVSMC. These findings suggest potential attractiveness of ILK inhibition to reduce established PH in males and suggest that the combination with estrogen-lowering drugs could be considered to maximize anti-proliferative and anti-remodeling effects of ILK inhibitors in females.

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.

Sex differences in hemodynamic responses and long-term survival to optimal medical therapy in patients with pulmonary arterial hypertension

It is widely known that the incidence of pulmonary arterial hypertension (PAH) is higher in female, whereas prognosis is poorer in male patients. However, sex differences in hemodynamic response to and long-term prognosis with PAH-targeted treatment in the modern era remain to be fully elucidated. We examined the long-term prognosis of 129 consecutive PAH patients (34 males and 95 females) diagnosed in our hospital from April 1999 to October 2014, and assessed hemodynamic changes in response to PAH-targeted therapy. Female patients had better 5-year survival compared with male patients (74.0 vs. 53.4%, P = 0.003); however, higher age quartiles in females were associated with poor outcome. Follow-up examination after medical treatment showed significant decreases in mean pulmonary arterial pressure (mPAP), pulmonary vascular resistance (PVR), and pulmonary arterial capacitance (PAC) in both sexes (both P < 0.05), whereas only females had a significant improvement in right ventricular end-diastolic pressure (RVEDP), right atrial pressure (RAP), cardiac index, and mixed venous oxygen saturation (SvO2) (all P < 0.05). Baseline age significantly correlated with the hemodynamic changes only in female patients; particularly, there were significant sex interactions in RVEDP and RAP (both P < 0.10). The multivariable analysis showed that SvO2 at baseline and mPAP and SvO2 at follow-up were significant prognostic factors in males, whereas the changes in mPAP, PVR, and PAC and use of endothelin-receptor antagonist in females. These results indicate that female PAH patients have better long-term prognosis than males, for which better improvements of right ventricular functions and hemodynamics may be involved.

Estrogen Preserves Pulsatile Pulmonary Arterial Hemodynamics in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is caused by extensive pulmonary vascular remodeling that increases right ventricular (RV) afterload and leads to RV failure. PAH predominantly affects women; paradoxically, female PAH patients have better outcomes than men. The roles of estrogen in PAH remain controversial, which is referred to as "the estrogen paradox". Here, we sought to determine the role of estrogen in pulsatile pulmonary arterial hemodynamic changes and its impact on RV functional adaption to PAH. Female mice were ovariectomized and replenished with estrogen or placebo. PAH was induced with SU5416 and chronic hypoxia. In vivo hemodynamic measurements showed that (1) estrogen prevented loss of pulmonary vascular compliance with limited effects on the increase of pulmonary vascular resistance in PAH; (2) estrogen attenuated increases in wave reflections in PAH and limited its adverse effects on PA systolic and pulse pressures; and (3) estrogen maintained the total hydraulic power and preserved transpulmonary vascular efficiency in PAH. This study demonstrates that estrogen preserves pulmonary vascular compliance independent of pulmonary vascular resistance, which provides a mechanical mechanism for ability of estrogen to delay disease progression without preventing onset. The estrogenic protection of pulsatile pulmonary hemodynamics underscores the therapeutic potential of estrogen in PAH.

17β-Estradiol Attenuates Conduit Pulmonary Artery Mechanical Property Changes With Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH), a rapidly fatal vascular disease, strikes women more often than men. Paradoxically, female PAH patients have better prognosis and survival rates than males. The female sex hormone 17β-estradiol has been linked to the better outcome of PAH in females; however, the mechanisms by which 17β-estradiol alters PAH progression and outcomes remain unclear. Because proximal pulmonary arterial (PA) stiffness, one hallmark of PAH, is a powerful predictor of mortality and morbidity, we hypothesized that 17β-estradiol attenuates PAH-induced changes in mechanical properties in conduit proximal PAs, which imparts hemodynamic and energetic benefits to right ventricular function. To test this hypothesis, female mice were ovariectomized and treated with 17β-estradiol or placebo. PAH was induced in mice using SU5416 and chronic hypoxia. Extra-lobar left PAs were isolated and mechanically tested ex vivo to study both static and frequency-dependent mechanical behaviors in the presence or absence of smooth muscle cell activation. Our static mechanical test showed significant stiffening of large PAs with PAH (P<0.05). 17β-Estradiol restored PA compliance to control levels. The dynamic mechanical test demonstrated that 17β-estradiol protected the arterial wall from the PAH-induced frequency-dependent decline in dynamic stiffness and loss of viscosity with PAH (P<0.05). As demonstrated by the in vivo measurement of PA hemodynamics via right ventricular catheterization, modulation by 17β-estradiol of mechanical proximal PAs reduced pulsatile loading, which contributed to improved ventricular-vascular coupling. This study provides a mechanical mechanism for delayed disease progression and better outcome in female PAH patients and underscores the therapeutic potential of 17β-estradiol in PAH.

Progress in solving the sex hormone paradox in pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a devastating and progressive disease with marked morbidity and mortality. Even though being female represents one of the most powerful risk factors for PAH, multiple questions about the underlying mechanisms remain, and two "estrogen paradoxes" in PAH exist. First, it is puzzling why estrogens have been found to be protective in various animal models of PAH, whereas PAH registries uniformly demonstrate a female susceptibility to the disease. Second, despite the pronounced tendency for the disease to develop in women, female PAH patients exhibit better survival than men. Recent mechanistic studies in classical and in novel animal models of PAH, as well as recent studies in PAH patients, have significantly advanced the field. In particular, it is now accepted that estrogen metabolism and receptor signaling, as well as estrogen interactions with key pathways in PAH development, appear to be potent disease modifiers. A better understanding of these interactions may lead to novel PAH therapies. It is the purpose of this review to 1) review sex hormone synthesis, metabolism, and receptor physiology; 2) assess the context in which sex hormones affect PAH pathogenesis; 3) provide a potential explanation for the observed estrogen paradoxes and gender differences in PAH; and 4) identify knowledge gaps and future research opportunities. Because the majority of published studies investigated 17β-estradiol and/or its metabolites, this review will primarily focus on pulmonary vascular and right ventricular effects of estrogens. Data for other sex hormones will be discussed very briefly.

A novel break point of the BMPR2 gene exonic deletion in a patient with pulmonary arterial hypertension

The presence of genetic rearrangements of bone morphogenetic protein type 2 receptor (BMPR2) was identified in pulmonary arterial hypertension (PAH) patients as the deletion or duplication of one or more exons of the gene. We recently investigated the deletion break points in exonic deletions of BMPR2 in two Japanese familial cases with PAH, and found that these were Alu-mediated via either non-allelic homologous recombination or non-homologous recombination. We herein report the third case of exonic deletion, which was in a 25-year-old female PAH patient with a deletion of BMPR2 exon 3. The break point in this case was not located in an Alu sequence. The 5'- and 3'-break point maps between the inverted Alu sequences in intron 2 and in exon 3, respectively, resulted in a 759-bp deletion. This novel exonic deletion in this PAH case may be a unique and non-recurrent rearrangement, and appears to be of a different size from that in other patients.

Absence of influence of gender and BMPR2 mutation type on clinical phenotypes of pulmonary arterial hypertension

BackgroundPrevious studies indicate that patients with pulmonary arterial hypertension (PAH) carrying a mutation in the bone morphogenetic protein receptor type 2 (BMPR2) gene, develop the disease 10 years earlier than non-carriers, and have a more severe hemodynamic compromise at diagnosis. A recent report has suggested that this may only be the case for females and that patients with missense mutations in BMPR2 gene have more severe disease than patients with truncating mutations.MethodsWe reviewed data from all patients with PAH considered as idiopathic and patients with a family history of PAH, who underwent genetic counselling in the French PAH network between January, 1st 2004 and April, 1st 2010. We compared clinical, functional, and hemodynamic characteristics between carriers and non-carriers of a BMPR2 mutation, according to gender or BMPR2 mutation type.ResultsPAH patients carrying a BMPR2 mutation (n = 115) were significantly younger at diagnosis than non-carriers (n = 267) (35.8 ± 15.4 and 47.5 ± 16.2 respectively, p < 0.0001). The presence of a BMPR2 mutation was associated with a younger age at diagnosis in females (36.4 ± 14.9 in BMPR2 mutation carriers and 47.4 ± 15.8 in non-carriers, p < 0.0001), and males (34.6 ± 16.8 in BMPR2 mutation carriers and 47.8 ± 17.1 in non-carriers, p < 0.0001). BMPR2 mutation carriers had a more severe hemodynamic compromise at diagnosis, but this was not influenced by gender. No differences in survival and time to death or lung transplantation were found in male and female PAH patients carrying a BMPR2 mutation. No differences were observed in clinical outcomes according to the type of BMPR2 mutations (missense, truncating, large rearrangement or splice defect).ConclusionWhen compared to non-carriers, BMPR2 mutation carriers from the French PAH network are younger at diagnosis and present with a more severe hemodynamic compromise, irrespective of gender. Moreover, BMPR2 mutation type had no influence on clinical phenotypes in our patient population.

Gene expression in BMPR2 mutation carriers with and without evidence of Pulmonary Arterial Hypertension suggests pathways relevant to disease penetrance

BackgroundWhile BMPR2 mutation strongly predisposes to pulmonary arterial hypertension (PAH), only 20% of mutation carriers develop clinical disease. This finding suggests that modifier genes contribute to FPAH clinical expression. Since modifiers are likely to be common alleles, this problem is not tractable by traditional genetic approaches. Furthermore, examination of gene expression is complicated by confounding effects attributable to drugs and the disease process itself.MethodsTo resolve these problems, B-cells were isolated, EBV-immortalized, and cultured from familial PAH patients with BMPR2 mutations, mutation positive but disease-free family members, and family members without mutation. This allows examination of differences in gene expression without drug or disease-related effects. These differences were assayed by Affymetrix array, with follow-up by quantitative RT-PCR and additional statistical analyses.ResultsBy gene array, we found consistent alterations in multiple pathways with known relationship to PAH, including actin organization, immune function, calcium balance, growth, and apoptosis. Selected genes were verified by quantitative RT-PCR using a larger sample set. One of these, CYP1B1, had tenfold lower expression than control groups in female but not male PAH patients. Analysis of overrepresented gene ontology groups suggests that risk of disease correlates with alterations in pathways more strongly than with any specific gene within those pathways.ConclusionDisease status in BMPR2 mutation carriers was correlated with alterations in proliferation, GTP signaling, and stress response pathway expression. The estrogen metabolizing gene CYP1B1 is a strong candidate as a modifier gene in female PAH patients.

Inpatients with congenital heart disease complicated with pulmonary arterial hypertension: an analysis of patient composition and clinical features

Objective:To investigate the composition and clinical features of inpatients with congenital heart disease (CHD) complicated with pulmonary arterial hypertension (PAH) from a cardiovascular department.Methods: We retrospectively analyzed the clinical data of 238 PAH patients who were identified from 3 674 CHD inpatients in Changhai Hospital during Jan, 2001 to Jul, 2007.Results: Among the 3 674 CHD inpatients who were treated in our hospital in recent 6 years, 238 (6.48%) had PAH.PAH was found in 10.8% of patients with atrial septal defect (ASD), 2.81% of patients with ventricular septal defects (VSD), 5.57% of patients with patent ductus arteriosus (PDA), and 19.6% of patients with complex types.The mean age of patients was (45.6±17.7) years, with 49.6% of them being 40-60 years.Female patients accounted for 69.3% of the total.The composition of PAH patients was: ASD (62.6%), VSD(19.7%), and PDA(13.4%).The patients with VSD,PDA or other complex types were younger than those with ASD(P0.01).More female PAH patients had ASD than VSD (76.5% vs 44.7%,P0.01).Most patients had chest distress and dyspnea on exertion as initial symptoms and accentuated pulmonary component of the second heart sound as physical sign; they also had increased cardiothoracic ratio, prominent, dilated central pulmonary arteries on chest radiography, right ventricle enlargement, and incomplete right bundle branch block on ECG.The pulmonary artery systolic pressure(sPAP), as estimated by echocardiography, had a significant correlation with catheterization measurements(r=0.734,P0.01).Conclusion: PAH is common in patients with CHD, especially in females, those with ASD, and those of 40-60 years old.The clinical presentation, chest radiography and ECG are normal.Echocardiography has important diagnosis value.There is a correlation between echocardiography and catheterization measurements of sPAP.