Familial Pulmonary Arterial Hypertension Research Articles

TASK-1 (KCNK3) channels in the lung: from cell biology to clinical implications.

TWIK-related acid-sensitive potassium channel 1 (TASK-1 encoded by KCNK3) belongs to the family of two-pore domain potassium channels. This gene subfamily is constitutively active at physiological resting membrane potentials in excitable cells, including smooth muscle cells, and has been particularly linked to the human pulmonary circulation. TASK-1 channels are sensitive to a wide array of physiological and pharmacological mediators that affect their activity such as unsaturated fatty acids, extracellular pH, hypoxia, anaesthetics and intracellular signalling pathways. Recent studies show that modulation of TASK-1 channels, either directly or indirectly by targeting their regulatory mechanisms, has the potential to control pulmonary arterial tone in humans. Furthermore, mutations in KCNK3 have been identified as a rare cause of both familial and idiopathic pulmonary arterial hypertension. This review summarises our current state of knowledge of the functional role of TASK-1 channels in the pulmonary circulation in health and disease, with special emphasis on current advancements in the field.

Genetics of Pulmonary Arterial Hypertension.

Tremendous progress has been made in understanding the genetics of pulmonary arterial hypertension (PAH) since its description in the 1950s as a primary disorder of the pulmonary vasculature. Heterozygous germline mutations in the gene coding bone morphogenetic receptor type 2 (BMPR2) are detectable in the majority of cases of heritable PAH, and in approximately 20% of cases of idiopathic pulmonary arterial hypertension (IPAH). However, recent advances in gene discovery methods have facilitated the discovery of additional genes with mutations among those with and without familial PAH. Heritable PAH is an autosomal dominant disease characterized by reduced penetrance, variable expressivity, and female predominance. Biallelic germline mutations in the gene EIF2AK4 are now associated with pulmonary veno-occlusive disease and pulmonary capillary hemangiomatosis. Growing genetic knowledge enhances our capacity to pursue and provide genetic counseling, although the issue remains complex given that the majority of carriers of PAH-related mutations will never be diagnosed with the disease.

The Impact of Heterozygous KCNK3 Mutations Associated With Pulmonary Arterial Hypertension on Channel Function and Pharmacological Recovery.

BackgroundHeterozygous loss of function mutations in the KCNK3 gene cause hereditary pulmonary arterial hypertension (PAH). KCNK3 encodes an acid‐sensitive potassium channel, which contributes to the resting potential of human pulmonary artery smooth muscle cells. KCNK3 is widely expressed in the body, and dimerizes with other KCNK3 subunits, or the closely related, acid‐sensitive KCNK9 channel.Methods and ResultsWe engineered homomeric and heterodimeric mutant and nonmutant KCNK3 channels associated with PAH. Using whole‐cell patch‐clamp electrophysiology in human pulmonary artery smooth muscle and COS7 cell lines, we determined that homomeric and heterodimeric mutant channels in heterozygous KCNK3 conditions lead to mutation‐specific severity of channel dysfunction. Both wildtype and mutant KCNK3 channels were activated by ONO‐RS‐082 (10 μmol/L), causing cell hyperpolarization. We observed robust gene expression of KCNK3 in healthy and familial PAH patient lungs, but no quantifiable expression of KCNK9, and demonstrated in functional studies that KCNK9 minimizes the impact of select KCNK3 mutations when the 2 channel subunits co‐assemble.ConclusionsHeterozygous KCNK3 mutations in PAH lead to variable loss of channel function via distinct mechanisms. Homomeric and heterodimeric mutant KCNK3 channels represent novel therapeutic substrates in PAH. Pharmacological and pH‐dependent activation of wildtype and mutant KCNK3 channels in pulmonary artery smooth muscle cells leads to membrane hyperpolarization. Co‐assembly of KCNK3 with KCNK9 subunits may provide protection against KCNK3 loss of function in tissues where both KCNK9 and KCNK3 are expressed, contributing to the lung‐specific phenotype observed clinically in patients with PAH because of KCNK3 mutations.

A burden of rare variants in BMPR2 and KCNK3 contributes to a risk of familial pulmonary arterial hypertension

BackgroundPulmonary arterial hypertension (PAH) is a severe lung disease with only few effective treatments available. Familial cases of PAH are usually recognized as an autosomal dominant disease, but incomplete penetrance of the disease makes it difficult to identify pathogenic variants in accordance with a Mendelian pattern of inheritance.MethodsTo elucidate the complex genetic basis of PAH, we obtained whole exome- or genome-sequencing data of 17 subjects from 9 families with heritable PAH and applied gene-based association analysis with 9 index patients and 300 PAH-free controls.ResultsA burden of rare variants in BMPR2 significantly contributed to the risk of the disease (p = 6.0 × 10−8). Eight of nine families carried four previously reported single nucleotide variants and four novel insertion/deletion variants in the gene. One of the novel variants was a large 6.5 kilobase-deletion. In the remaining one family, the patient carried a pathogenic variant in a member of potassium channels, KCNK3, which was the first replicative finding of channelopathy in an Asian population.ConclusionsThe variety of rare pathogenic variants suggests that gene-based association analysis using genome-wide sequencing data from increased number of samples is essential to tracing the genetic heterogeneity and developing an appropriate panel for genetic testing.

Modulation of Endothelial Bone Morphogenetic Protein Receptor Type 2 Activity by Vascular Endothelial Growth Factor Receptor 3 in Pulmonary Arterial Hypertension.

Bone morphogenetic protein (BMP) signaling has multiple roles in the development and function of the blood vessels. In humans, mutations in BMP receptor type 2 (BMPR2), a key component of BMP signaling, have been identified in the majority of patients with familial pulmonary arterial hypertension (PAH). However, only a small subset of individuals with BMPR2 mutation develops PAH, suggesting that additional modifiers of BMPR2 function play an important role in the onset and progression of PAH. We used a combination of studies in zebrafish embryos and genetically engineered mice lacking endothelial expression of Vegfr3 to determine the interaction between vascular endothelial growth factor receptor 3 (VEGFR3) and BMPR2. Additional in vitro studies were performed by using human endothelial cells, including primary lung endothelial cells from subjects with PAH. Attenuation of Vegfr3 in zebrafish embryos abrogated Bmp2b-induced ectopic angiogenesis. Endothelial cells with disrupted VEGFR3 expression failed to respond to exogenous BMP stimulation. Mechanistically, VEGFR3 is physically associated with BMPR2 and facilitates ligand-induced endocytosis of BMPR2 to promote phosphorylation of SMADs and transcription of ID genes. Conditional, endothelial-specific deletion of Vegfr3 in mice resulted in impaired BMP signaling responses, and significantly worsened hypoxia-induced pulmonary hypertension. Consistent with these data, we found significant decrease in VEGFR3 expression in pulmonary arterial endothelial cells from human PAH subjects, and reconstitution of VEGFR3 expression in PAH pulmonary arterial endothelial cells restored BMP signaling responses. Our findings identify VEGFR3 as a key regulator of endothelial BMPR2 signaling and a potential determinant of PAH penetrance in humans.

Patient-Specific iPSC-Derived Endothelial Cells Uncover Pathways that Protect against Pulmonary Hypertension in BMPR2 Mutation Carriers

In familial pulmonary arterial hypertension (FPAH), the autosomal dominant disease-causing BMPR2 mutation is only 20% penetrant, suggesting that genetic variation provides modifiers that alleviate the disease.Here, we used comparison of induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) from three families with unaffected mutation carriers (UMCs), FPAH patients, and gender-matched controls to investigate this variation. Our analysis identified features of UMC iPSC-ECs related to modifiers of BMPR2 signaling or to differentially expressed genes. FPAH-iPSC-ECs showed reduced adhesion, survival, migration, and angiogenesis compared to UMC-iPSC-ECs and control cells. The "rescued" phenotype of UMC cells was related to an increase in specific BMPR2 activators and/or a reduction in inhibitors, and the improved cell adhesion could be attributed to preservation of related signaling. The improved survival was related to increased BIRC3 and was independent of BMPR2. Our findings therefore highlight protective modifiers for FPAH that could help inform development of future treatment strategies.

Genetic analyses in a cohort of children with pulmonary hypertension.

The prevalence of germline mutations in paediatric pulmonary hypertension (PH) is poorly documented. The objective of this study was to determine the mutation frequency in PH genes in a paediatric cohort and describe the clinical characteristics of mutation carriers.The study involved 66 index cases with PH: 35 children with idiopathic pulmonary arterial hypertension (IPAH); five children with familial PAH (FPAH); three children with pulmonary veno-occlusive disease (PVOD); and 23 children with PAH associated with congenital heart disease (APAH-CHD).No mutations were found in the 23 children with APAH-CHD. In the 40 children with IPAH or FPAH, 12 mutations were found: five on BMPR2; four on ACVRL1; and three on TBX4. In the three PVOD cases, two carried the EIF2AK4 mutation. Mutation carriers had a more severe disease at diagnosis and more aggressive first-line therapy was required. The three patients with PVOD had a very severe disease at diagnosis and required a lung transplantation.The genetic architecture of paediatric PAH is enriched in ACVRL1 and TBX4 mutations compared to adult PAH, but further studies are required to confirm these results. Childhood-onset PAH in children carrying a mutation in one of the genes tested has a more severe presentation at diagnosis but a similar outcome to that observed in non-carriers.

Bone Morphogenetic Protein Receptor Type 2 Mutation in Pulmonary Arterial Hypertension: A View on the Right Ventricle.

The effect of a mutation in the bone morphogenetic protein receptor 2 (BMPR2) gene on right ventricular (RV) pressure overload in patients with pulmonary arterial hypertension is unknown. Therefore, we investigated RV function in patients who have pulmonary arterial hypertension with and without the BMPR2 mutation by combining in vivo measurements with molecular and histological analysis of human RV and left ventricular tissue. In total, 95 patients with idiopathic or familial pulmonary arterial hypertension were genetically screened for the presence of a BMPR2 mutation: 28 patients had a BMPR2 mutation, and 67 patients did not have a BMPR2 mutation. In vivo measurements were assessed using right heart catheterization and cardiac MRI. Despite a similar mean pulmonary artery pressure (noncarriers 54±15 versus mutation carriers 55±9 mm Hg) and pulmonary vascular resistance (755 [483-1043] versus 931 [624-1311] dynes·s(-1)·cm(-5)), mutation carriers presented with a more severely compromised RV function (RV ejection fraction: 37.6±12.8% versus 29.0±9%: P<0.05; cardiac index 2.7±0.9 versus 2.2±0.4 L·min(-1)·m(-2)). Differences continued to exist after treatment. To investigate the role of transforming growth factor β and bone morphogenetic protein receptor II signaling, human RV and left ventricular tissue were studied in controls (n=6), mutation carriers (n=5), and noncarriers (n=11). However, transforming growth factor β and bone morphogenetic protein receptor II signaling, and hypertrophy, apoptosis, fibrosis, capillary density, inflammation, and cardiac metabolism, as well, were similar between mutation carriers and noncarriers. Despite a similar afterload, RV function is more severely affected in mutation carriers than in noncarriers. However, these differences cannot be explained by a differential transforming growth factor β, bone morphogenetic protein receptor II signaling, or cardiac adaptation.

Abstract 148: Blockade of the Serotonin 2B Receptor Prevents Pulmonary Vascular Stiffening and Remodeling in a Mouse Model of Familial Pulmonary Arterial Hypertension

Objective: Pulmonary arterial hypertension (PAH) is a fatal disease of the pulmonary vasculature. In humans, familial PAH is caused by mutations in the bone morphogenetic protein receptor 2 gene (BMPR2). Serotonin signaling through the 2B receptor (5HT2BR) is also important in the pathogenesis of PAH, making 5HT2BR antagonism a potential target for PAH therapeutics. This study evaluates the hypothesis that antagonism of 5HT2BR will prevent PAH in a genetic mouse model and the pulmonary vascular stiffening and remodeling that is considered the underlying cause. Methods: Rosa26-Bmpr2 R899X mice on an FVB/N background express the patient-derived R899X mutation in BMPR2 in all tissues when induced with doxycycline. 10-14 week old BMPR2 mutant mice were fed doxycycline in a high-fat diet for 6 weeks. After 2 weeks, osmotic pumps containing either the 5HT2BR antagonist SB204741 (1 mg/kg/day) or DMSO/water vehicle were implanted. Right ventricular systolic pressure (RVSP) was measured via a venous jugular catheter under anesthesia, and the mice euthanized. Lungs were collected and prepared for histology, gene expression analysis, and atomic force microscopy (AFM, Bioscope Catalyst). Immortalized pulmonary microvascular smooth muscle cells (SMCs) with a BMPR2 mutation were assessed for long-term changes in cytoskeletal contractility using a free floating collagen gel assay. Data: BMPR2 R899X mice treated with SB204741 failed to develop elevated RVSP. Histologic analysis revealed that SB204741 reduced both the total number of inflammatory cells in the lung as well as the muscularization of peripheral pulmonary arterioles. Microarray analysis revealed that 5HT2BR antagonism primarily changed the expression contractility-associated genes. Measurements of pulmonary arteriole elastic modulus with AFM show significantly increased vessel wall stiffness in BMPR2 R899X arterioles that is also normalized with SB204741 treatment. Taken together, these data suggest that 5HT2BR antagonism prevents the vascular stiffening and remodeling that causes PAH.

BMPR2 gene delivery reduces mutation-related PAH and counteracts TGF-β-mediated pulmonary cell signalling.

Idiopathic, familial and secondary pulmonary arterial hypertension (PAH) are associated with reduced bone morphogenetic protein receptor type 2 (BMPR2) expression, and in some contexts, TGF-β upregulation. Our aims were to assess BMPR2 gene therapy in a PAH mouse model and to assess the impact on TGF-β signalling. Using a targeted in vivo gene delivery approach, we assessed the impact of BMPR2 gene delivery in a transgenic mouse model in which PAH was first induced by doxycycline driven expression of a dominant negative BMPR2 mutant (R899X). We also assessed the impact of BMPR2 gene delivery on TGF-β-induced changes in cell signalling in human pulmonary vascular endothelial and smooth muscle cells. In the mouse model, changes in TGF-β levels were not detected, but BMPR2 gene delivery reversed the increase in right ventricle systolic pressure (RVSP) and Fulton Index (FI), associated with a trend to increased pulmonary endothelial nitric oxide synthase (eNOS) gene expression. In vitro, BMPR2 gene transfer reduced TGF-β effects on Smad2, Smad1/5/8 and Erk1/2 phosphorylation in human pulmonary arterial smooth muscle cells (HPASMC). BMPR2 was also found to upregulate nitric oxide (NO) production in lung derived human microvascular endothelial cells (HMVEC-L). This study provides further evidence that BMPR2 modulation may have therapeutic potential. See Editorial, page 406.

Abstract 17231: Contact-Mediated Interaction Between Pulmonary Artery Endothelial and Smooth Muscle Cells Promotes a BMPR2-β-catenin-Notch1 Signal Causing Hyperpolarization of Endothelial Mitochondria and a Stalk Cell-like Phenotype

Background: Pulmonary arterial hypertension (PAH) is characterized by endothelial (EC) apoptosis related to loss of distal (D) pulmonary arteries (PA) and abnormal proliferation of smooth muscle-like cells (SMC). These features are mediated by impaired cell metabolism. We observed mitochondrial dysfunction in human PAEC with reduced or mutant BMPR2, the gene associated with familial PAH. Deletion of BMPR2 in murine EC causes failure to regenerate DPA and to recover from hypoxia induced pulmonary hypertension. Loss of BMPR2 in PASMC results in mitochondrial dysfunction associated with enhanced proliferation. Since metabolites and other factors are often transferred by cell contact, we investigated how PASMC-PAEC contact impacts PAEC mitochondrial and cellular function, and could be perturbed by a BMPR2 mutation in PAEC and/or PASMC. Methods and Results: We used a co-culture system in which PAEC and PASMC from lungs of donor control (c-) or PAH patients with a BMPR2 mutation (m-) were either seeded on opposite sides of a 1μm pore insert or separated to assess non-contact mediated responses. When c-PAEC contacted c-PASMC, PAEC mitochondrial membrane potential (Δψ) increased 6-fold compared to mono-cultured or non-contact co-cultured cells (p &lt; 0.001). These PAEC exhibited a significant increase in proliferation (3-fold increase by Ki-67 staining, p&lt;0.01) and survival (73% reduction in caspase activity, p &lt; 0.001); however we observed a decrease in migration and angiogenic capacity in modified Boyden chamber and matrigel assays (52% and 60% reduction, p &lt; 0.001, respectively). We related the increase in Δψ and the above ‘stalk cell-like’ phenotype to an increase in Notch1 intracellular domain (2.5-fold increase, p &lt; 0.001), as they were abrogated by the gamma secretase inhibitor, and enhanced by the co-transcriptional activator β-catenin (4-fold increase, p &lt; 0.001). When m-PAEC and m-PASMC were used in contact cultures, these features were not observed. Conclusions: PASMC in contact with PAEC increase Δψ and produce a Notch and β-catenin mediated stalk cell-like EC phenotype. These features are lost when BMPR2 is deficient in both PAEC and PASMC suggesting that BMPR2-mediated PASMC-EC contact allows PAEC to survive and regenerate in response to damage.

Abstract 12841: Mutations in the BMPR2 Gene Are Associated With Worse Survival in Patients With Pulmonary Arterial Hypertension

Introduction: Mutations in the BMPR2 gene are the commonest genetic cause of pulmonary arterial hypertension (PAH), however the impact of BMPR2 mutations on clinical phenotype and outcomes remains uncertain. Methods: We collated individual participant-level data on 1550 patients with idiopathic, familial and anorexigen-associated PAH from 8 studies that had systematically tested for the presence of BMPR2 mutations. 1164 patients from 6 studies were included in the primary survival analysis. Associations of BMPR2 mutation status with risk of all cause death and death or transplant were assessed by calculating age and sex adjusted hazard ratios (95% CI) using Cox proportional-hazard regression models stratified by study. Results: Overall, 448 (28.9%) of 1550 patients had a BMPR2 mutation. BMPR2 mutation carriers were younger at diagnosis, (mean ±SD) 35 ±15 versus 42 ±18 years, p&lt;0.001), had a higher mean pulmonary artery pressure (61 ±14 versus 56 ±15 mmHg, p&lt;0.001) and pulmonary vascular resistance (17 ±8 versus 13 ±8 Wood units, p&lt;0.001) and lower cardiac index (2.1 ±0.7 versus 2.5 ±0.9 L/min/m2, p&lt;0.001) at diagnostic right heart catheterization. Patients with BMPR2 mutations were less likely to respond to acute vasodilator testing (2.7% versus 16.6%, p&lt;0.001). Hazard ratios for those carrying a BMPR2 mutation for all cause death and death or transplant are shown in the figure below. In a complete case analysis, the increased risk of all cause death (HR 1.50 (1.07-2.12), p=0.02) and death or transplant (HR 1.76 (1.31-2.38), p&lt;0.001) in those aged &lt;50 at diagnosis persisted after adjusting for right atrial pressure, pulmonary vascular resistance and NYHA functional class at diagnosis. Conclusions: Patients with PAH and BMPR2 mutations are younger at diagnosis and present with more severe disease. Patients with BMPR2 mutations (particularly those diagnosed at a younger age) are at increased risk of death or transplantation compared with BMPR2 negative patients.

Abstract 19602: Characterization of Tgfβ/BMP-axis in Multiple Animal Models of PH

Introduction: Mutations in the bone morphogenetic protein recptor type 2 (BMPR2) comprise a large portion of familial Pulmonary Arterial Hypertension (PAH) cases. The transforming growth factor-β (TGF-β)/BMP-axis in PAH has been of interest, which is hypothesized to favor TGF-β signaling due to defective BMPR2 signaling, consequently leading to pro-proliferative signaling within the lung. In addition, it has been proposed that the BMPRII mutations might affect cardiac adaptation. To date none of the available animal models have been fully characterized with regard to the TGF-β/BMP pathway. This study assessed the lung and heart TGF-β/BMP-axis in multiple rat animal models of pulmonary hypertension to ensure translational capability. Methods: Heart and lung TGF-β/BMP-axis was assessed by qPCR, western blot and immunofluorescence in the the monocrotaline (MCT), Sugen-hypoxia (SuHx), Sugen-Pneumonectomy (SuPnx) and Pulmonary artery banding model (PAB) and compared to control and PAH patient tissues. Circulating ligands, TGF-β receptor (TGFβR) type 1 and 2 and BMPR2, and canonical downstream signaling (Smad2/3, Smad1/5/8, and transcription factors) were investigated. Results: BMPR2 was down-regulated at both transcription and protein levels in the lung of all PH animal models (p&lt;0.05). Transcription of pulmonary TGFβR1 and -2 were increased in the SuPNx-model, compared to control (P&lt;0.001). In both SuHx and SuPnx models an increase in protein Smad2/3 expression was observed by immunofluorescence implying overactivation of TGF-β signaling. Cardiac TGFβR1 was decreased in PAB model, compared to control (P&lt;0.05), while TGFβR2 was decreased in both the MCT and PAB model. Conclusion: Early indications reveal differences between several pulmonary hypertension animal models, with regard to the TGF-β/BMP pathway. Additional analysis is needed to fully characterize the regulation of TGF-β and BMP in the rat models.

New Formulae for Calculating Left Heart Pressure in Pulmonary Artery Hypertension Patients at the Bedside

Objective: To compare new empirical formulae in HeartSmart, to calculate the mean pulmonary artery and mean pulmonary artery occlusion (wedge) pressures to that of the Pulmonary Artery Catheter Thermodilution Method. Method: 20 adult patients with pulmonary artery hypertension admitted on to Bradford Royal Infirmary NHS Trust Hospital UK Intensive Care Unit, where haemodynamic monitoring is routinely performed as part of the treatment using the thermodilution method compared to the HeartSmart to guide interventional fluid resuscitation etc. Each subject has six paired values of each left heart pressure and cardiac indexes to test the robustness of HeartSmart against the perceived ‘gold standard’ thermodilution method. This was a double blind study the haemodynamic variables of each technology were close enough to each other. Results: Bland-Altman plot comparing the measured mean pulmonary artery pressure (MPAP) mmHg to the Heartsmart calculated pulmonary artery pressures (CPAP) mmHg: For mean of the differences between MPAP mmHg and CPAP mmHg = - 1.08 mmHg, Legend 1 . Comparing the measured mean pulmonary artery occlusion (wedge) pressure (MPWP) mmHg to the HeartSmart calculated pulmonary artery occlusion (wedge) pressure (CPWP) mmHg: For mean of the differences between MPWP mmHg and CPWP mmHg = - 0.56 mmHg, Legend 2 . Comparing the measured mean cardiac index in litres/min/m 2 MCI to the HeartSmart calculated mean cardiac index in litres/min/m 2 CCI: For mean of the differences between MCI litres/min/m 2 and CCI litres/min/m 2 = -0.03 litres/min/m 2 ; Legend 5 . Conclusion: The results demonstrate HeartSmart TM software compares favourably with the Pulmonary Artery Catheter thermodilution method at the bedside and could be used interchangeably or replace PACTD. Background: The incidence of pulmonary artery hypetension is on the increase in the United Kingdom. Primary pulmonary arterial hypertension is a rare disease that is characterized by increased pulmonary-artery pressure in the absence of common secondary causes of pulmonary hypertension, such as viruses auto immune system, chronic heart, lung, or thromboembolic disease. Before the advent of new and some novel therapies, patients with idiopathic or familial pulmonary arterial hypertension average survival rates were less than 7 years. Despite the progress in diagnostic techniques and treatment, pulmonary arterial hypertension is becoming more common although remaining a progressive, debilitating fatal disease. The clinical signs and symptoms can be nonspecific, patients often receive a diagnosis too late as the condition advances, deteriorates and of course treatment is less effective. The use of the (PAC) right heart floatation pulmonary artery catheter (Swan-Ganz) to measure the mean pulmonary artery and mean pulmonary artery occlusion (capillary wedge) pressures is one means of confirming the diagnosis of pulmonary artery hypertension, although its use is not appropriate in neonates or paediatric cases.

Sequencing of mutations in the serine/threonine kinase domain of the bone morphogenetic protein receptor type 2 gene causing pulmonary arterial hypertension.

Objective:Germline mutations in the bone morphogenetic protein receptor type-2 (BMPR2) gene are considered to be a major risk factor for pulmonary arterial hypertension (PAH). BMPR2 mutations have been reported in 10%–20% of idiopathic PAH and in 80% of familial PAH cases. The aim of this study was to evaluate the frequency of mutations in the serine/threonine kinase domain of the BMPR2 gene in a group of patients from a single PAH referral center in Turkey.Methods:This cross-sectional study used a DNA-sequencing method to investigate BMPR2 mutations in the serine-threonine-kinase domain in 43 patients diagnosed with PAH [8 with idiopathic PAH and 35 with congenital heart disease (CHD)] from a single PAH referral center. Patients were included if they had a hemodynamically measured mean pulmonary arterial pressure of >25 mm Hg with a mean pulmonary capillary wedge pressure of ≤15 mm Hg. Patients with severe left heart disease and/or pulmonary disease that could cause pulmonary hypertension were excluded. Associations between categoric variables were determined using the chi-square test. Differences between idiopathic and CHD-associated PAH groups were compared with the unpaired Student’s t-test for continuous variables.Results:We detected a missense mutation, [p.C347Y (c.1040G>A)], in one patient with idiopathic PAH in exon 8 of the BMPR2 gene. The mutation was detected in a 27-year-old female with a remarkable family history for PAH. She had a favorable response to endothelin receptor antagonists. No mutations were detected in the exons 5–11 of the BMPR2 gene in the PAH-CHD group.Conclusion:A missense mutation was detected in only one of the eight patients with idiopathic PAH. The BMPR2 missense mutation rate of 12.5% in this cohort of Turkish patients with idiopathic PAH was similar to that seen in European registries. The index patient was a young female with a family history remarkable for PAH; she had a good long-term response to PAH-specific treatment, probably due to the early initiation of the treatment. Genetic screening of families affected by PAH might have great value in identifying the disease at an early stage.

Validation of two predictive models for survival in pulmonary arterial hypertension.

The French Pulmonary Hypertension Network (FPHN) registry and the Registry to Evaluate Early And Long-term Pulmonary Arterial Hypertension Disease Management (REVEAL) have developed predictive models for survival in pulmonary arterial hypertension (PAH). In this collaboration, we assess the external validity (or generalisability) of the FPHN ItinérAIR-HTAP predictive equation and the REVEAL risk score calculator. Validation cohorts approximated the eligibility criteria defined for each model. The REVEAL cohort comprised 292 treatment-naïve, adult patients diagnosed <1 year prior to enrolment with idiopathic, familial or anorexigen-induced PAH. The FPHN cohort comprised 1737 patients with group 1 PAH. Application of FPHN parameters to REVEAL and REVEAL risk scores to FPHN demonstrated estimated hazard ratios that were consistent between studies and had high probabilities of concordance (hazard ratios of 0.72, 95% CI 0.64-0.80, and 0.73, 95% CI 0.70-0.77, respectively). The REVEAL risk score calculator and FPHN ItinérAIR-HTAP predictive equation showed good discrimination and calibration for prediction of survival in the FPHN and REVEAL cohorts, respectively, suggesting prognostic generalisability in geographically different PAH populations. Once prospectively validated, these may become valuable tools in clinical practice.

Blood pressure variability and cardiovascular risk in 9 eastern european countries: data from the BP-Care study

Methods: Total 327 patients were enrolled from 2008 to 2011 in the Korean registry program. They met the criteria of PAH and we investigated the clinical data, the medication prescribed and survival status et al. Results: Their mean age was 40.8 15.8years with female /male patient ratio of 2.7:1. The patients in WHO I/II were 32.4% and WHO III/IV were 67.6%. The peak/mean pulmonary arterial pressure was 86.3/ 56.6 mmHg. Idiopathic PAH (22%), connective tissue disease (24%) and congenital heart disease (46%) were predominant. Small number of patients with familial PAH, portal hypertension, HIV infection, pulmonary capillary hemangiomatosis (PCH) existed. The patients described bosentan were 107, sildenafil 50, iloprost 31, and veraprost 49. After follow-up for mean ( SD) duration of 37.2 18.1 months of idiopathic PAH, the survival rates at 1, 2, and 3 years were 70.0%, 64%, and 55% respectively. The survival rates of patients with non-idiopathic PAH were better than that of idiopathic PAH patients. Conclusions: The baseline characteristics and survival rates of our cohort study are close to those of the National Institutes of Health Registry of other countries, and the survival rates of patients with idiopathic PAH were worse than that of non-idiopathic PAH patients. Therefore, the target related therapy should be more popular for the improvement of survival rate of idiopathic PAH patients.

Defective cellular trafficking of the bone morphogenetic protein receptor type II by mutations underlying familial pulmonary arterial hypertension

Familial pulmonary arterial hypertension (FPAH) is a relatively rare but fatal disorder characterized by elevated arterial pressure caused by abnormal proliferation of endothelial cells of the arteries, which eventually leads to heart failure and death. FPAH is inherited as an autosomal dominant trait and is caused by heterozygous mutations in the BMPR2 gene encoding the bone morphogenetic protein type II receptor (BMPR2). BMPR2 belongs to the TGF β/BMP super-family of receptors involved in a signal transduction cascade via the SMAD signaling pathway. The BMPR2 polypeptide is composed of 1038 amino acids and consists of a ligand binding domain, a kinase domain and a cytoplasmic tail. To investigate the cellular and functional consequence of BMPR2 mutations, C-terminally FLAG-tagged constructs of eighteen pathogenic BMPR2 missense mutants were generated by site directed mutagenesis and expressed in HeLa and HEK-293T cell lines. The subcellular localizations of the mutant proteins were investigated using immunostaining and confocal microscopy. Post-translational modifications of the proteins were analyzed by Endoglycosidase H deglycosylation assay. Our results indicated that mutations in the ligand binding domain affecting highly conserved cysteine residues resulted in retention of the mutant proteins in the endoplasmic reticulum (ER), as evident from their co-localization with the ER resident protein calnexin. The kinase domain mutants showed both ER and plasma membrane (PM) distributions, while the cytoplasmic tail domain variants were localized exclusively to the PM. The subcellular localizations of the mutants were further confirmed by their characteristic glycosylation profiles. In conclusion, our results indicate that ER quality control (ERQC) is involved in the pathological mechanism of several BMPR2 receptor missense mutations causing FPAH, which can be explored as a potential therapeutic target in the future.

Endothelial-to-mesenchymal transition in pulmonary hypertension.

The vascular remodeling responsible for pulmonary arterial hypertension (PAH) involves predominantly the accumulation of α-smooth muscle actin-expressing mesenchymal-like cells in obstructive pulmonary vascular lesions. Endothelial-to-mesenchymal transition (EndoMT) may be a source of those α-smooth muscle actin-expressing cells. In situ evidence of EndoMT in human PAH was obtained by using confocal microscopy of multiple fluorescent stainings at the arterial level, and by using transmission electron microscopy and correlative light and electron microscopy at the ultrastructural level. Findings were confirmed by in vitro analyses of human PAH and control cultured pulmonary artery endothelial cells. In addition, the mRNA and protein signature of EndoMT was recognized at the arterial and lung level by quantitative real-time polymerase chain reaction and Western blot analyses. We confirmed our human observations in established animal models of pulmonary hypertension (monocrotaline and SuHx). After establishing the first genetically modified rat model linked to BMPR2 mutations (BMPR2(Δ140Ex1/+) rats), we demonstrated that EndoMT is linked to alterations in signaling of BMPR2, a gene that is mutated in 70% of cases of familial PAH and in 10% to 40% of cases of idiopathic PAH. We identified molecular actors of this pathological transition, including twist overexpression and vimentin phosphorylation. We demonstrated that rapamycin partially reversed the protein expression patterns of EndoMT, improved experimental PAH, and decreased the migration of human pulmonary artery endothelial cells, providing the proof of concept that EndoMT is druggable. EndoMT is linked to alterations in BPMR2 signaling and is involved in the occlusive vas cular remodeling of PAH, findings that may have therapeutic implications.

Microvessel mechanobiology in pulmonary arterial hypertension: cause and effect.

Pulmonary arterial hypertension (PAH, or group 1 pulmonary hypertension) is a progressive, insidious, and fatal illness of the pulmonary microvasculature. Proliferative remodeling of the small resistance arteries results in an elevation of mean pulmonary arterial pressure (mPAP) and total pulmonary vascular resistance (PVR), increasing the workload on the right ventricle (RV) until it eventually fails.1 Although these hemodynamic parameters are useful for clinical diagnosis, alone they have limited prognostic value; furthermore, vasodilator therapy aimed at reduction of PVR has achieved only modest success, and increases in patient survival are attributed primarily to earlier diagnosis and intervention.2 These limitations have motivated research focused on identifying measurable physiological parameters that could both predict mortality and serve as measures for therapeutic efficacy. Decreased pulmonary arteriolar compliance (PAC) is a major factor contributing to the increased RV workload and failure in PAH.3–5 PAC measures a vessel’s ability to deform under loading, and as a blood vessel stiffens its compliance decreases. Total vessel compliance is estimated as stroke volume divided by pulse pressure (PP). This estimation alone is a strong predictor of survival in idiopathic and familial PAH.5 Although proximal artery stiffness has received a great deal of attention in hypoxic pulmonary hypertension and is important in increasing RV workload, changes in PAC affect the entire pulmonary vasculature with the largest portion of that change occurring in vessels distal to the lung hilum.6 Understanding how vascular remodeling changes PAC, especially in the distal vasculature where much of our knowledge on vessel mechanics is currently lacking, will allow us to link the cellular and molecular mediators commonly studied in PAH with the biomechanical changes that cause elevated pressures and RV failure. Pulmonary arterial smooth muscle cells (PASMCs) and adventitial fibroblasts decrease PAC by altering the composition, amount, and organization …