Heritable Pulmonary Arterial Hypertension Research Articles

Heritable pulmonary arterial hypertension

Heritable pulmonary arterial hypertension (PAH) can be triggered by at least 18genes. The most frequently altered gene is the bone morphogenetic protein receptor2 (BMPR2). Further genes from the same pathway are also well known PAH-causing genes. Genetic testing can aid to confirm differential diagnoses such as apulmonary veno-occlusive disease. It also enables the testing of healthy family members. In addition to the PAH patient population particularly served by genetic testing, this article touches on the mode of inheritance and provides insights into the first treatments soon on the market that rebalance the BMPR2 signaling pathway.

Coexistence of pulmonary arterial hypertension and straight back syndrome in a patient with a novel BMPR2 variant affecting cytoplasmic tail domain

BackgroundPathologic variants in the bone morphogenetic protein receptor-2 (BMPR2) gene cause a pulmonary arterial hypertension phenotype in an autosomal-dominant pattern with incomplete penetrance. Straight back syndrome is one of the causes of pseudo-heart diseases. To date, no cases of idiopathic or heritable pulmonary arterial hypertension with straight back syndrome have been reported.Case presentationA 30-year-old female was diagnosed with pulmonary arterial hypertension by right heart catheterization. Computed tomography revealed a decreased anteroposterior thoracic space with heart compression, indicating a straight back syndrome. Genetic analysis by whole exome sequencing identified a novel c.2423_2424delGT (p.G808Gfs*4) germline frameshift variant within BMPR2 affecting the cytoplasmic tail domain.ConclusionsThis is the first report of different straight back characteristics in heritable pulmonary arterial hypertension with a novel germline BMPR2 variant. This finding may provide a new perspective on the variable penetrance of the pulmonary arterial hypertension phenotype.

Pathobiology of Pulmonary Arterial Hypertension

Abstract: Pulmonary Arterial Hypertension (PAH) is a progressive disease associated with occlusive pulmonary arterial remodeling of vessels < 500 μm for which there is no cure. Even in the era of PAH-specific combination therapies, aberrant lung pathology and progressive right ventricular (RV) dysfunction occur, culminating in a median survival of 6.2 years, according to the latest data in the treatment era. While better than a median survival from symptom onset of 2.8 years prior to PAH-specific therapies, it is still poor. Thus, there is an urgent need to move the opportunities forward for meaningful treatment strategies. Clearly, a better understanding of the highly complex pathobiology of PAH is needed if we are to achieve new and novel treatment strategies. This is especially so if we are to pursue a more personalized treatment approach to PAH in light of the multitude of pathobiological abnormalities described in PAH, which likely culminate in a final common pathway for PAH development. In this State-of-the-Art review, we provide comprehensive insights into the complex pathobiology of PAH to provide understanding and insights for the practicing clinician. We review the pathology of PAH and the cells involved and their impact in driving pathological abnormalities (pulmonary artery endothelial cells, smooth muscle cells, fibroblasts and pericytes) as well as the role of the extracellular matrix. Inflammation and immune dysfunction are considered important drivers of PAH and are comprehensively discussed. Another pathway relates to TGFβ/ bone morphogenic protein (BMP) imbalance, which is highlighted, as well as a new novel agent, sotatercept that impacts this imbalance. Genetic factors underlying heritable PAH (HPAH) are addressed, as well as epigenetic influences. Other important pathways highlighted include growth factor signaling, ion channels/channelopathy, hypoxia signaling pathways, and altered metabolism and mitochondrial dysfunction. We also address the “estrogen paradox”, whereby PAH is more common in women but more severe in men. The basis for drug-induced PAH is discussed, including the new methamphetamine epidemic. We briefly provide insights into DNA damage and senescence factors in pathobiology and highlight commonalities between PAH and cancer pathobiology. Furthermore, we provide concluding insights for the treating physician. In conclusion, we need to pose the right questions to motivate novel and effective treatment strategies for the management of PAH based on pathobiological principles and understanding.

Hemodynamic and Clinical Profiles of Pulmonary Arterial Hypertension Patients with GDF2 and BMPR2 Variants.

Asians have a higher carrier rate of pulmonary arterial hypertension (PAH)-related genetic variants than Caucasians do. This study aimed to identify PAH-related genetic variants using whole exome sequencing (WES) in Asian idiopathic and heritable PAH cohorts. A WES library was constructed, and candidate variants were further validated by polymerase chain reaction and Sanger sequencing in the PAH cohort. In a total of 69 patients, the highest incidence of variants was found in the BMPR2, ATP13A3, and GDF2 genes. Regarding the BMPR2 gene variants, there were two nonsense variants (c.994C>T, p. Arg332*; c.1750C>T, p. Arg584*), one missense variant (c.1478C>T, p. Thr493Ile), and one novel in-frame deletion variant (c.877_888del, p. Leu293_Ser296del). Regarding the GDF2 variants, there was one likely pathogenic nonsense variant (c.259C>T, p. Gln87*) and two missense variants (c.1207G>A, p. Val403Ile; c.38T>C, p. Leu13Pro). The BMPR2 and GDF2 variant subgroups had worse hemodynamics. Moreover, the GDF2 variant patients were younger and had a significantly lower GDF2 value (135.6 ± 36.2 pg/mL, p = 0.002) in comparison to the value in the non-BMPR2/non-GDF2 mutant group (267.8 ± 185.8 pg/mL). The BMPR2 variant carriers had worse hemodynamics compared to the patients with the non-BMPR2/non-GDF2 mutant group. Moreover, there was a significantly lower GDF2 value in the GDF2 variant carriers compared to the control group. GDF2 may be a protective or corrected modifier in certain genetic backgrounds.

Lung Transplantation in Pulmonary Arterial Hypertension: The Portuguese Experience

In patients with pulmonary arterial hypertension (PAH), refractory to medical therapy, lung transplantation emerges as an option. This study describes the outcomes of 8 PAH patients who underwent lung transplantation. A retrospective, single-center study was conducted among patients with PAH who underwent lung transplantation in our center. Patients had a median age of 46 years, with female sex predominance (75%). Causes of HAP were pulmonary veno-occlusive disease (n = 5, 62.5%), idiopathic PAH (n = 2, 25%), and heritable PAH (n = 1, 12.5%). Pre-transplant hemodynamics revealed a median mean pulmonary artery pressure of 58.5 mm Hg (48-86). All patients received bilateral lung transplants with extracorporeal membrane oxygenation support, displaying immediate post-transplant hemodynamic improvement. Primary graft dysfunction grade 3 (PGD 3) was observed in 75% of patients. Five patients (62.5%) died, with a 72.9% survival at 12 months and 29.2% at 24 months post-transplantation. Our study reveals the complexity and challenges of lung transplants in patients with PAH. Despite notable immediate hemodynamic improvements, high rates of PGD 3 and the survival rate remain a concern. Further research to define optimal peri and post-transplant management to improve survival is required.

Long Term Survival of Heritable Pulmonary Arterial Hypertension Associated with Hereditary Hemorrhagic Telangiectasia: A Case Series.

Hereditary hemorrhagic telangiectasia (HHT) is a hereditary disease characterized by recurrent epistaxis, mucocutaneous telangiectasias, and visceral arteriovenous malformations. Multiple genetic mutations have been linked to this rare disease, including ENG, ALK1 (ACVRL1), and MADH4. Pulmonary hypertension is a potential complication of HHT, with the most common phenotypes being World Health Organization (WHO) group 1 heritable pulmonary arterial hypertension (PAH), which is typically associated with ALK1 mutation; WHO group 2 pulmonary hypertension due to high output heart failure from hepatic arteriovenous malformations and/or anemia; and WHO group 2 due to high pulmonary artery wedge pressure. There is scarce evidence to help guide treatment of heritable PAH in HHT, and observational literature suggests that patients with HHT and heritable PAH have a worse prognosis compared to patients with idiopathic PAH. We describe the diagnosis, pulmonary hemodynamics, and detailed treatment courses of three patients with ALK1-associated HHT and PAH, who all exhibited objective clinical improvement with parenteral prostacyclins and oral agents.

The prevalence and significance of the EIF2AK4 variants in the patients with pulmonary arterial hypertension

Abstract Background Eukaryotic translation initiation factor 2 alpha kinase 4 (EIF2AK4) has been reported to be the causative gene of pulmonary veno-occlusive disease (PVOD).[1] Although some patients with pulmonary arterial hypertension (PAH) also carry the EIF2AK4 variants, its importance remains unclear. Purpose The purpose of this study was to evaluate the prevalence and significance of EIF2AK4 variants in patients with PAH. Methods The whole exome sequencing were performed in total 162 patients with idiopathic or heritable PAH. The clinical data including the high-resolution CT scanning, pulmonary function test, laboratory data at diagnosis, and therapeutic response to pulmonary vasodilator were evaluated. Results We identified the heterozygous variants of EIF2AK4 in 6 (3.7%) patients. Among them, 2 patients also had bone morphogenetic protein receptor type 2 (BMPR2) variants which were defined as pathogenic by American College of Medical Genetics and Genomics classification. The other 4 patients did not have known PAH associated gene variants. Although the typical findings of PVOD such as interlobular septal thickening and enlarged mediastinal lymph nodes, were not identified by the CT scanning, the median percent diffusing capacity for carbon monoxide of patients with EIF2AK4 heterozygous variants was reduced to 45.6%. In addition, 4 of 6 patients received prostacyclin injection therapy because oral combination therapy was ineffective, and 2 patients died. Moreover, none of the 6 patients reached mean pulmonary arterial pressure≤30 mmHg despite the combination therapy. We also identified homozygous EIF2AK4 variants in 2 patients with PAH. They did not have the known causative variants of PAH. CT scanning at diagnosis did not detect any characteristic of PVOD in either of the two patients. Although the pulmonary vasodilator was initiated without induction of pulmonary edema, one patient was refractory to the therapy. Conclusions The genetic analysis is crucial for accurate diagnosis even in patients diagnosed with PAH because heterozygous or homozygous variants in EIF2AK4 could be involved in the disease pathology.

Pulmonary arterial hypertension associated to toxic-oil syndrome. A long story short

Abstract Introduction Pulmonary arterial hypertension (PAH) is occasionally related to previous use of drugs or toxins. The toxic oil syndrome (TOS) is a model of drug-induced PAH caused by the prior use of denatured rapeseed oil. This rare condition caused an acute epidemic disease in Spain, including the development of pulmonary arterial hypertension, with a mortality rate of 82% from 1981 to 1987. Several cases have been discovered in the successive years until 2018. The clinical profile and outcome of those patients with PAH-TOS diagnosed since 1988 are unknown. Methods Historical cases of PAH-TOS since 1988 were collected. We analysed baseline characteristics, genetic testing, and the rates of lung transplantation or mortality in PAH-TOS, comparing these outcomes with incident idiopathic and heritable PAH cases (i/hPAH) from the Spanish Registry of Pulmonary Hypertension (REHAP). Results Thirty-nine patients with PAH-TOS between 1988 and 2018 were selected for this study. The median time between the toxic intake and the disease onset was 20.3 years (Interquartile range: 14.3 – 25.6). At least one patient per year was identified in that period, with a mean incidence of 1.63 cases/per year. Genetic testing was done in 21 patients, using a 37-gene Next Generation Sequencing panel, not finding any pathogenic or likely pathogenic variant. Importantly, in 3 out of 39 cases, the disease resolved (7.7%), while 4 cases needed lung transplantation. The 10-year mortality was 35.9%. Cases of PAH-TOS were compared with 200 incident patients with i/hPAH collected from 2003 onwards. PAH-TOS patients were significantly younger when compared with i/hPAH patients (p<0.0035). At baseline, patients with PAH-TOS had higher haemodynamically severity when compared with i/hPAH (mean pulmonary artery pressure of 65.0 mmHg Vs 53.5 mmHg, and pulmonary vascular resistance of 15.1 Vs 12.1 Wood Units, respectively; p=0.003, and p=0.02). In PAH-TOS the 5- and 10-year survival free of lung transplantation was 81.3%, and 52.7%, similar to the rates in the i/hPAH group (76.6%, and 59.2%, respectively; Log-rank test p=0.881. Figure). Conclusions The toxic oil syndrome is a unique model of drug-induced PAH, with new cases diagnosed several years after the toxic exposure. We did not find genetic variants in PAH-SOT patients. As described in other drug-induced cases of PAH, some patients experience a resolution of the disease over time. The very long-term survival of these patients is similar to that of patients with idiopathic or heritable pulmonary arterial hypertension.Figure

Abstract 14665: Cybrid Analysis Delineates Nuclear and Mitochondrial Contributions to Oxidative Stress in Pulmonary Arterial Hypertension

Rationale : Heritable pulmonary arterial hypertension (HPAH) is a serious vasculopathy caused by remodeling of pulmonary arterioles. It is inherited in an autosomal dominant pattern with reduced penetrance, suggesting additional triggers are needed for disease development. Past study shows increased DNA damage and total reactive oxygen species (ROS) in lymphoblastoid cells (LCL) from PAH patients and unaffected relatives, indicating a potential genetic component. Hypothesis: Increased oxidative stress is an independent genetically-determined modifier of PAH susceptibility. Methods: LCL from patients, relatives and unrelated controls were analyzed for total and mitochondrial (mt) ROS and mt function (Seahorse). Untargeted metabolomics were performed by Metabolon. Nuclear vs mt gene contribution was studied using cybrids created by transferring the mitochondria from high (HC) and low ROS LCL (LC) to mt negative 143B Rho0 cells. Results: Metabolomics revealed significant differences between PAH and control LCL in the TCA cycle and glutathione metabolism relating to oxidative stress. HPAH LCL showed significant upregulation of both ROS species compared to controls. LCL from relatives clustered into two groups, one with increased mtROS, the other comparable to controls . Like PAH lung vascular cells, LCL with increased mtROS had reduced spare respiratory capacity (SRC) suggesting dysfunctional mitochondria, but no glycolytic switch. Cybrid analysis revealed that HCs had significantly higher basal respiration (p=0.02), with SRC trending higher compared to LCs, indicating a functional electron transport chain. These cells also had elevated mtROS (p=0.04), suggesting reduced antioxidants. Conclusions: HPAH is a complex disease, and mutation status alone doesn’t determine its susceptibility. Patient LCL recapitulate some of the metabolomic pathways dysregulated in lung vascular cells. Oxidative stress in LCL extends to some unaffected relatives, suggesting this is an independent genetic trait that modifies PAH risk. Our cybrid analysis, novel in PAH highlights the importance of nuclear-mt crosstalk. WGS is underway to identify nuclear variants that could affect the mt, and act as potential modifiers and second hits in PAH pathogenesis.

Abstract 18578: BMP9 Regulates Endothelial Expression of Mediators of Pulmonary Vascular Remodeling

Background: Loss-of-function mutations in GDF2 /BMP9 are found in heritable pulmonary arterial hypertension (PAH), suggesting a protective role, while protective or pathogenic effects are found in experimental PAH studies depending on context. The net contribution of BMP9 in PAH thus remains controversial. Hypothesis: BMP9 regulates the secretome of pulmonary microvascular endothelium (PMVEC) to contribute to pulmonary artery smooth muscle cell (PASMC) phenotype modulation. Methods: We tested recombinant BMP9, BMP9/BMP10 trap ALK1-Fc, and BMP9 neutralizing antibody before and after development of pulmonary hypertension (PH) in distinct experimental models. We analyzed the impact of BMP9 on the PMVEC transcriptome, intersection with human and experimental PAH lung transcriptomes, and tested the impact of EC-derived genes on PASMC phenotype and growth. Results: ALK1-Fc and anti-BMP9 prevented or reversed experimental PH administered before or after onset of experimental PH. Recombinant disulfide-linked BMP9 was not protective in SUGEN/Hypoxia (SU-Hx)- or monocrotaline (MCT)-induced experimental PH, in contrast to previous studies using incompletely disulfide-linked BMP9, suggesting dissociated monomers might function as competitive BMP9 antagonists. Consistent with this interpretation, BMP9 upregulated PMVEC genes associated with pulmonary vascular remodeling in experimental and human PAH including CXCL12, IGFBP4, ET-1, VEGF-A, and PDGF-BB. Stimulation of PMVEC with BMP9 induced a contractile phenotype ( CNN1;TAGLN ) in co-cultured PASMC. Administration of ALK1-Fc or anti-BMP9 attenuated expression of several of these growth factors in SU-Hx PH. In experimental PH ALK1-Fc and ACTRIIA-Fc elicited peripheral edema, a phenotype associated with use of ALK1-Fc in human trials. Conclusions: BMP9 contributes to experimental PH by regulating expression of EC-derived molecules modulating PASMC growth and phenotype. In contrast to broad strategies that inhibit BMP9 and BMP10 associated with HHT mimicry in clinical trials and rodent PH models, treatment with anti-BMP9 was well-tolerated. Selective antagonism of BMP9 represents a potential therapeutic strategy for human PAH.

Abstract 17276: NOTCH3-BMPR Interactions Drive Smooth Muscle Cell Proliferation and Medial Hyperplasia in Pulmonary Arterial Hypertension

Background: One of the characteristics of pulmonary arterial hypertension (PAH) is the proliferation of smooth muscle cells. In heritable PAH, aberrant BMPR2 signaling results in low levels of intracellular ID1. An important pathway involved in vascular smooth muscle cell proliferation and the development of non-heritable PAH is the NOTCH3-HES5 signaling pathway. Previous studies have demonstrated that HES-5 and ID-1 are peptides belonging to the basic helix-loop-helix family, and they act as transcriptional enhancers of cell cycle genes. Transcriptional enhancement or repression requires the heterodimerization of different HES and ID proteins. Hypothesis: We hypothesize that HES-5 binds to and degrades IDs in non-heritable cases of PAH. Methods: The half-life of ID proteins was assessed in human small pulmonary artery smooth muscle cells that either constitutively expressed or minimally expressed HES-5. In order to understand whether this effect was due to direct binding between HES-5 and ID1, co-immunoprecipitation experiments were performed. Immunofluorescent staining was used to detect the expression level of ID1 and HES-5. Proximity ligation assay (PLA) was utilized to examine of endogenous in-situ interaction of ID1-HES5. Results: We found that overexpression of HES-5 significantly diminished the half-life of ID1 and ID3, and had little effect on ID2. All three proteins localized to the nucleus of small pulmonary artery smooth muscle cells. Immunofluorescent staining confirmed elevated levels of HES and reduced levels of ID1 and ID3 in the medial layer of PAH vessels. By PLA assay, we show that HES-5 and ID1 as well as HES-5 and ID-3 physically associate with each other to the level of 40 nanometers. Conclusions: Constitutive NOTCH3 signaling and high intracellular HES-5 levels drive vascular smooth muscle cell proliferation in non-familial PAH by binding to and degrading ID intracellular proteins. Our study demonstrates cross-talk between the NOTCH3-pathway and the BMPR2 pathway in this disease, at the level of their downstream effectors: ID1 and HES-5. Further studies should shed light on how these two pathways control the PAH disease process.

445 Awardee Talk: Generation of a BMPR2 Heterozygous Ovine Genetic Model for Heritable Pulmonary Arterial Hypertension

Abstract Pulmonary Arterial Hypertension (PAH) is a rare vascular disorder affecting 1 to 2 cases per million individuals. Monoallelic mutations in the BMPR2 gene are the most common genetic risk factor, with about 20% of carriers affected. BMPR2 is a serine/threonine kinase receptor in the transforming growth factor beta (TGF-β) superfamily. Despite well-known associations with PAH, the role of BMPR2 in the progression of the disorder remains poorly understood due in part to the lack of appropriate preclinical genetic models. The homozygous BMPR2 (-/-) knockout genotype is embryonic lethal, which is an obstacle to creating a suitable genetic biomedical model for PAH. To overcome this barrier, we used a single guide RNA (sgRNA) CRISPR/Cas9 approach to target exon 3 of the ovine BMPR2 gene, in combination with a single-stranded oligo-deoxynucleotide (ssODN) synonymous homology-directed repair (HDR) template to create heterozygous BMPR2 (+/-) sheep. Heterozygote lambs could be generated in two ways with this approach – high editing efficiency coupled with ssODN homology-directed repair of one of the edited alleles, OR intermediate editing efficiency with low to no presence of HDR in the genetic sequence. Our main objectives were to:1.) Determine optimal concentrations of ssODN (100, 300, and 600 ng/ul) to promote HDR and 2.) Identify the sgRNA which maximized the probability of obtaining heterozygous lambs. A ssODN was designed to serve as a synonymous repair template that disrupted the PAM site for a pair of sgRNAs targeting BMPR2 exon 3 on opposite strands. We electroporated ovine zygotes six hours post-fertilization using sgRNA (100 ng/µL)/Cas9 (200 ng/µL) ribonucleoprotein (RNP) complexes alongside three ODN concentrations (100, 300, and 600 ng/µL). Zygotes were cultured for seven days, and blastocysts were lysed, PCR-amplified, and Sanger sequenced. Sequences were analyzed using TIDER (Brinkman et al., Nucl. Acids Res., 2018). TIDER is a computer model which quantifies the frequency of targeted small nucleotide changes introduced by CRISPR in combination with HDR using a donor template. A Kruskal-Wallis test compared the efficacy of HDR at 100, 300, and 600 ng/µL of ODN template. A significant difference was found between groups with 600 ng/µL giving the highest rate of HDR (P < 0.0001). Post-hoc pairwise comparisons using a Dunn's test found that sgRNA1 at 600 ng/ul ODN was significantly more efficient for HDR repair as compared with sgRNA2 (P < 0.05). The editing efficiency of the blastocysts collected using the 600 ng/µLODN and sgRNA1 treatment combination was 44.7% high (>66%), 42.6% intermediate (66-33%), and 8.5% low (< 33%) knockout rate, and 4.2% wild-type. The knockin rate was 27.7% high (>20%), 21.3% intermediate (20-5%), and 51% low to no (< 5%) HDR. These data predict that about one-half the transferred embryos subject to these conditions will have the desired heterozygous genotype [BMPR2 (+/-) sheep].

Treprostinil Effectiveness in Higher-Risk Pediatric Patients With Idiopathic and Heritable Pulmonary Arterial Hypertension

BackgroundLittle is known about the effectiveness of treprostinil in higher-risk paediatric patients with various pulmonary arterial hypertension genotypes. This study was designed to investigate the prognosis of higher-risk paediatric patients with idiopathic or heritable pulmonary arterial hypertension (IPAH/HPAH) after treprostinil therapy. MethodsChildren with IPAH/HPAH who were stratified as higher risk and treated with treprostinil in our centre were included as the study cohort. Those who received only oral medications were included as the reference cohort. All patients in the study cohort received PAH-related genotyping. Survival was defined as no death. Event-free survival was defined as no death, Potts shunt, or atrial septostomy. ResultsForty-nine children (median age 7.7 years [interquartile range (IQR) 4.2-11.5 years], 65% female) were included in the study cohort and 48 children were included in the reference cohort; 84% of the study cohort had genetic disorders after genetic testing with a dominance of BMPR2 and ACVRL1 mutations. After a median therapy duration of 5.56 months (IQR 2.66-11.12 months), all patients were alive with significant improvements in clinical characteristics. One-, 2-, and 3-year survival rates were 91%, 84%, and 69%, respectively with a median follow-up duration of 19.17 months (IQR 9.7-29.79 months), which was significantly superior to the reference cohort (P = 0.038). Multivariate Cox regression analysis identified World Health Organisation functional class after therapy as a predictor for survival. There was no significant difference in survival among patients with different genotypes. ConclusionsTreprostinil can significantly improve the prognosis in children with IPAH/HPAH who are at higher risk, despite genetic backgrounds.

Pulmonary hypertension associated with diazoxide: the SUR1 paradox.

The ATP-sensitive potassium channels and their regulatory subunits, sulfonylurea receptor 1 (SUR1/Kir6.2) and SUR2/Kir6.1, contribute to the pathophysiology of pulmonary hypertension (PH). Loss-of-function pathogenic variants in the ABCC8 gene, which encodes for SUR1, have been associated with heritable pulmonary arterial hypertension. Conversely, activation of SUR1 and SUR2 leads to the relaxation of pulmonary arteries and reduces cell proliferation and migration. Diazoxide, a SUR1 activator, has been shown to alleviate experimental PH, suggesting its potential as a therapeutic option. However, there are paradoxical reports of diazoxide-induced PH in infants. This review explores the role of SUR1/2 in the pathophysiology of PH and the contradictory effects of diazoxide on the pulmonary vascular bed. Additionally, we conducted a comprehensive literature review of cases of diazoxide-associated PH and analysed data from the World Health Organization pharmacovigilance database (VigiBase). Significant disproportionality signals link diazoxide to PH, while no other SUR activators have been connected with pulmonary vascular disease. Diazoxide-associated PH seems to be dose-dependent and potentially related to acute effects on the pulmonary vascular bed. Further research is required to decipher the differing pulmonary vascular consequences of diazoxide in different age populations and experimental models.

Pulmonary arterial hypertension drugs can partially restore altered angiogenic capacities in bmpr2-silenced human lung microvascular endothelial cells.

Mutations in the bone morphogenetic protein receptor type 2 (bmpr2) gene and signaling pathway impairment are observed in heritable and idiopathic pulmonary arterial hypertension (PAH). In PAH, endothelial dysfunction is currently handled by drugs targeting the endothelin-1 (ET-1), nitric oxide (NO), and prostacyclin (PGI2) pathways. The role of angiogenesis in the disease process and the effect of PAH therapies on dysregulated angiogenesis remain inconclusive. We aim to investigate in vitro whether (i) bmpr2 silencing can impair angiogenic capacity of human lung microvascular endothelial cells (HLMVECs) and (ii) PAH therapies can restore them. The effects of macitentan (ET-1), tadalafil (NO), and selexipag (PGI2), on BMPRII pathway activation, endothelial barrier function, and angiogenesis were investigated in bmpr2-silenced HLMVECs. Stable bmpr2 silencing resulted in impaired migration and tube formation in vitro capacity. Inhibition of ET-1 pathway was able to partially restore tube formation in bmpr2-silenced HLMVECs, whereas none of the therapies was able to restore endothelial barrier function, no deleterious effects were observed. Our findings highlight the potential role of BMPRII signaling pathway in driving pulmonary endothelial cell angiogenesis. In addition, PAH drugs display limited effects on endothelial function when BMPRII is impaired, suggesting that innovative therapeutic strategies targeting BMPRII signaling are needed to better rescue endothelial dysfunction in PAH.

Bilateral lung transplantation for pediatric pulmonary arterial hypertension: perioperative management and one-year follow-up.

Bilateral lung transplantation (LuTx) remains the only established treatment for children with end-stage pulmonary arterial hypertension (PAH). Although PAH is the second most common indication for LuTx, little is known about optimal perioperative management and midterm clinical outcomes. Prospective observational study on consecutive children with PAH who underwent LuTx with scheduled postoperative VA-ECMO support at Hannover Medical School from December 2013 to June 2020. Twelve patients with PAH underwent LuTx (mean age 11.9 years; age range 1.9-17.8). Underlying diagnoses included idiopathic (n = 4) or heritable PAH (n = 4), PAH associated with congenital heart disease (n = 2), pulmonary veno-occlusive disease (n = 1), and pulmonary capillary hemangiomatosis (n = 1). The mean waiting time was 58.5 days (range 1-220d). Three patients were bridged to LuTx on VA-ECMO. Intraoperative VA-ECMO/cardiopulmonary bypass was applied and VA-ECMO was continued postoperatively in all patients (mean ECMO-duration 185 h; range 73-363 h; early extubation). The median postoperative ventilation time was 28 h (range 17-145 h). Echocardiographic conventional and strain analysis showed that 12 months after LuTx, all patients had normal biventricular systolic function. All PAH patients are alive 2 years after LuTx (median follow-up 53 months, range 26-104 months). LuTx in children with end-stage PAH resulted in excellent midterm outcomes (100% survival 2 years post-LuTx). Postoperative VA-ECMO facilitates early extubation with rapid gain of allograft function and sustained biventricular reverse-remodeling and systolic function after RV pressure unloading and LV volume loading.

Imaging Features in BMPR2 Mutation-associated Pulmonary Arterial Hypertension.

Background Germline mutation in the BMPR2 gene is common in patients with pulmonary arterial hypertension (PAH). However, its association with imaging findings in these patients is, to the knowledge of the authors, unknown. Purpose To characterize distinctive pulmonary vascular abnormalities at CT and pulmonary artery angiography in patients with and without BMPR2 mutation. Materials and Methods In this retrospective study, chest CT scans, pulmonary artery angiograms, and genetic test data were acquired for patients diagnosed with idiopathic PAH (IPAH) or heritable PAH (HPAH) between January 2010 and December 2021. Perivascular halo, neovascularity, centrilobular ground-glass opacity (GGO), and panlobular GGO were evaluated at CT and graded on a four-point severity scale by four independent readers. Clinical characteristics and imaging features between patients with BMPR2 mutation and noncarriers were analyzed using the Kendall rank-order coefficient and the Kruskal-Wallis test. Results This study included 82 patients with BMPR2 mutation (mean age, 38 years ± 15 [SD]; 34 men; 72 patients with IPAH and 10 patients with HPAH) and 193 patients without the mutation, all with IPAH (mean age, 41 years ± 15; 53 men). A total of 115 patients (42%; 115 of 275) had neovascularity, and 56 patients (20%; 56 of 275) had perivascular halo at CT, and so-called frost crystals were observed on pulmonary artery angiograms in 14 of 53 (26%) patients. Compared with patients without BMPR2 mutation, patients with BMPR2 mutation more frequently showed two distinctive radiographic manifestations, perivascular halo and neovascularity (38% [31 of 82] vs 13% [25 of 193] in perivascular halo [P < .001] and 60% [49 of 82] vs 34% [66 of 193] in neovascularity [P < .001], respectively). "Frost crystals" were more frequent in patients with BMPR2 mutation compared with noncarriers (53% [10 of 19] vs 12% [four of 34]; P < .01). Severe perivascular halo frequently coexisted with severe neovascularity in patients with BMPR2 mutation. Conclusion Patients with PAH with BMPR2 mutation showed distinctive features at CT, specifically perivascular halo and neovascularity. This suggested a link between the genetic, pulmonary, and systemic manifestations that underly the pathogenesis of PAH. © RSNA, 2023 Supplemental material is available for this article.

Serum and Pulmonary Expression Profiles of the Activin Signaling System in Pulmonary Arterial Hypertension.

Activins are novel therapeutic targets in pulmonary arterial hypertension (PAH). We therefore studied whether key members of the activin pathway could be used as PAH biomarkers. Serum levels of activin A, activin B, α-subunit of inhibin A and B proteins, and the antagonists follistatin and follistatin-like 3 (FSTL3) were measured in controls and in patients with newly diagnosed idiopathic, heritable, or anorexigen-associated PAH (n=80) at baseline and 3 to 4 months after treatment initiation. The primary outcome was death or lung transplantation. Expression patterns of the inhibin subunits, follistatin, FSTL3, Bambi, Cripto, and the activin receptors type I (ALK), type II (ACTRII), and betaglycan were analyzed in PAH and control lung tissues. Death or lung transplantation occurred in 26 of 80 patients (32.5%) over a median follow-up of 69 (interquartile range, 50-81) months. Both baseline (hazard ratio, 1.001 [95% CI, 1.000-1.001]; P=0.037 and 1.263 [95% CI, 1.049-1.520]; P=0.014, respectively) and follow-up (hazard ratio, 1.003 [95% CI, 1.001-1.005]; P=0.001 and 1.365 [95% CI, 1.185-1.573]; P<0.001, respectively) serum levels of activin A and FSTL3 were associated with transplant-free survival in a model adjusted for age and sex. Thresholds determined by receiver operating characteristic analyses were 393 pg/mL for activin A and 16.6 ng/mL for FSTL3. When adjusted with New York Heart Association functional class, 6-minute walk distance, and N-terminal pro-B-type natriuretic peptide, the hazard ratios for transplant-free survival for baseline activin A <393 pg/mL and FSTL3 <16.6 ng/mL were, respectively, 0.14 (95% CI, 0.03-0.61; P=0.009) and 0.17 (95% CI, 0.06-0.45; P<0.001), and for follow-up measures, 0.23 (95% CI, 0.07-0.78; P=0.019) and 0.27 (95% CI, 0.09-0.78, P=0.015), respectively. Prognostic values of activin A and FSTL3 were confirmed in an independent external validation cohort. Histological analyses showed a nuclear accumulation of the phosphorylated form of Smad2/3, higher immunoreactivities for ACTRIIB, ALK2, ALK4, ALK5, ALK7, Cripto, and FSTL3 in vascular endothelial and smooth muscle layers, and lower immunostaining for inhibin-α and follistatin. These findings offer new insights into the activin signaling system in PAH and show that activin A and FSTL3 are prognostic biomarkers for PAH.

SOX17 Enhancer Variants Disrupt Transcription Factor Binding And Enhancer Inactivity Drives Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is a rare disease characterized by remodeling of the pulmonary arteries, increased vascular resistance, and right-sided heart failure. Genome-wide association studies of idiopathic/heritable PAH established novel genetic risk variants, including conserved enhancers upstream of transcription factor (TF) SOX17 containing 2 independent signals. SOX17 is an important TF in embryonic development and in the homeostasis of pulmonary artery endothelial cells (hPAEC) in the adult. Rare pathogenic mutations in SOX17 cause heritable PAH. We hypothesized that PAH risk alleles in an enhancer region impair TF-binding upstream of SOX17, which in turn reduces SOX17 expression and contributes to disturbed endothelial cell function and PAH development. CRISPR manipulation and siRNA were used to modulate SOX17 expression. Electromobility shift assays were used to confirm in silico-predicted TF differential binding to the SOX17 variants. Functional assays in hPAECs were used to establish the biological consequences of SOX17 loss. In silico analysis with the connectivity map was used to predict compounds that rescue disturbed SOX17 signaling. Mice with deletion of the SOX17-signal 1 enhancer region (SOX17-4593/enhKO) were phenotyped in response to chronic hypoxia and SU5416/hypoxia. CRISPR inhibition of SOX17-signal 2 and deletion of SOX17-signal 1 specifically decreased SOX17 expression. Electromobility shift assays demonstrated differential binding of hPAEC nuclear proteins to the risk and nonrisk alleles from both SOX17 signals. Candidate TFs HOXA5 and ROR-α were identified through in silico analysis and antibody electromobility shift assays. Analysis of the hPAEC transcriptomes revealed alteration of PAH-relevant pathways on SOX17 silencing, including extracellular matrix regulation. SOX17 silencing in hPAECs resulted in increased apoptosis, proliferation, and disturbance of barrier function. With the use of the connectivity map, compounds were identified that reversed the SOX17-dysfunction transcriptomic signatures in hPAECs. SOX17 enhancer knockout in mice reduced lung SOX17 expression, resulting in more severe pulmonary vascular leak and hypoxia or SU5416/hypoxia-induced pulmonary hypertension. Common PAH risk variants upstream of the SOX17 promoter reduce endothelial SOX17 expression, at least in part, through differential binding of HOXA5 and ROR-α. Reduced SOX17 expression results in disturbed hPAEC function and PAH. Existing drug compounds can reverse the disturbed SOX17 pulmonary endothelial transcriptomic signature.

Is iron deficiency caused by BMPR2 mutations or dysfunction in pulmonary arterial hypertension patients?

AbstractIron deficiency is common in idiopathic and heritable pulmonary arterial hypertension patients (I/HPAH). A previous report suggested a dysregulation of the iron hormone hepcidin, which is controlled by BMP/SMAD signaling involving the bone morphogenetic protein receptor 2 (BMPR‐II). Pathogenic variants in the BMPR2 gene are the most common cause of HPAH. Their effect on patients' hepcidin levels has not been investigated. The aim of this study was to assess whether iron metabolism and regulation of the iron regulatory hormone hepcidin was disturbed in I/HPAH patients with and without a pathogenic variant in the gene BMPR2 compared to healthy controls. In this explorative, cross‐sectional study hepcidin serum levels were quantified by enzyme‐linked immunosorbent assay. We measured iron status, inflammatory parameters and hepcidin modifying proteins such as IL6, erythropoietin, and BMP2, BMP6 in addition to BMPR‐II protein and mRNA levels. Clinical routine parameters were correlated with hepcidin levels. In total 109 I/HPAH patients and controls, separated into three groups, 23 BMPR2 variant‐carriers, 56 BMPR2 noncarriers and 30 healthy controls were enrolled. Of these, 84% had iron deficiency requiring iron supplementation. Hepcidin levels were not different between groups and corresponded to the degree of iron deficiency. The levels of IL6, erythropoietin, BMP2, or BMP6 showed no correlation with hepcidin expression. Hence, iron homeostasis and hepcidin regulation was largely independent from these parameters. I/HPAH patients had a physiologically normal iron regulation and no false elevation of hepcidin levels. Iron deficiency was prevalent albeit independent of pathogenic variants in the BMPR2 gene.