2020 Annual World Congress of the Pulmonary Vascular Research Institute

Inhibition of MAP kinase-activated protein kinase 2 (MK2) prevents development of pulmonary hypertension

Mohammad Shafiq

Kashif Hanif

Division of Pharmacology, CSIR—Central Drug Research Institute, Lucknow, India

Pulmonary hypertension (PH), a fatal cardio-pulmonary disease, is characterized by pulmonary vascular remodeling, due to excessive proliferation of pulmonary vascular cells, and increased right ventricle pressure (RVP). Although a number of pathways are involved in pathophysiology of PH, there are increasing evidences which suggest an important role for inflammation in PH. Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2), a major downstream target of p38 MAPK, plays a pivotal role in inflammation. Therefore, we hypothesized that MK2 may contribute in pathophysiology of PH. For this, human pulmonary artery smooth muscle cells (HPASMCs) were exposed to hypoxia for 72 h. HPASMCs proliferated extensively and significantly expressed MK2. siRNA-mediated inhibition of MK2 significantly prevented the proliferation of HPASMCs, reduced expression of MK2, and decreased inflammatory markers (IL-1β and NF-ĸb). Furthermore, we confirmed the role of MK2 in PH by using MK2-deficient mice (MK2^–/–). Monocrotaline (MCT) (600 mg/kg, i.p.) was administered once a week for five weeks in wild-type and MK2^–/– mice. As compared to control mice, there was a significant rise in RVP in wild-type mice but no significant rise in RVP was observed in MK2^–/– mice. MCT-treated mice (wild type) also showed increased right ventricle (RV) mass and reduction in ejection fraction as compared to control mice, whereas MCT-treated MK2^–/– mice showed no significant increase in RV mass and reduction in ejection fraction. Furthermore, pharmacological inhibition of MK2 by MMI-0100 in MCT-treated PH rats reduced RVP, hypertrophy, RV mass, and improved ejection fractions and other cardiac functions. These results show that MK2 plays an important role in increasing inflammation and its inhibition may be protective in PH.

Sex differences in pediatric high altitude pulmonary edema

Dunbar Ivy, John T. Brinton, Maxene R. Meier, Jason P. Weinman and Deborah R. Liptzin

Departments of Pediatrics, Biostatistics and Informatics, and Radiology, University of Colorado, Aurora, CO, USA

To explore sex differences in pediatric high altitude pulmonary edema (HAPE). We performed a retrospective chart review in children who presented to Children’s Hospital Colorado from 2004 to 2014 with a clinical diagnosis consistent with HAPE and a chest radiograph consistent with non-cardiogenic pulmonary edema. Descriptive statistics were used to describe the demographics, presentations, treatment strategies, and follow-up. Results showed that from 2004 to 2014, 50 children met criteria for HAPE. Median age at presentation was 10.2 years (range 0.6–19 years). Thirty patients had classic HAPE, 19 patients had re-entry HAPE, and 1 had high altitude resident pulmonary edema. Most (72%) patients were male. This sex difference was most notable in children older than 11 years of age (p = 0.004) and was driven by children with classic HAPE (p = 0.007) rather than re-entry HAPE (p = 0.25). In conclusion, HAPE appears to be more common in post-pubescent males. Further studies should be done to confirm this sex difference, to determine if female sex hormones are protective against HAPE or male sex hormones are a risk factor for HAPE, and to explore underlying mechanisms to better inform treatment. (This study was supported by grant UL1 TR001082 from the NIH/NCRR Colorado CTSI; the Jayden de Luca Foundation; and the Frederick and Margaret Weyerhaeuser Foundation.)

Role of vascular mineralocorticoid receptors in pulmonary hypertension

Divya P. Menon, Guanming Qi, Seung K. Kim, M. Elizabeth Moss, Krishna C. Penumatsa, Rod R. Warburton, Deniz Toksoz, Jamie Wilson, Nicholas S. Hill, Iris Z. Jaffe and Ioana R. Preston

Pulmonary, Critical Care and Sleep Division, Molecular Cardiology Research Institute, Tufts Medical Center, Boston, MA, USA

Abnormalities that characterize pulmonary arterial hypertension (PAH) include an impairment in the structure and function of pulmonary vascular endothelial (EC) and smooth muscle cells (SMC). Levels of the hormone aldosterone are elevated in human PAH patients and in experimental pulmonary hypertension (PH). Inhibition of the aldosterone-binding mineralocorticoid receptor (MR) attenuates PH in multiple animal models. This study explored the specific role of MR in pulmonary vascular ECs and SMCs in PH using cell-specific MR knockout mice exposed to Sugen/hypoxia-induced PH. SMC-MR-KO mice, EC-MR-KO mice, and their appropriate controls were exposed to SU5416 (Su)/hypoxia or control normoxia. Some MR-KO mice and their controls were also treated with spironolactone (SP) from day 1 of hypoxia exposure. Hemodynamic measurements were obtained at the end of exposure. Right ventricular (RV) hypertrophy was expressed as the Fulton index (RV/LV + septum). Heart and lung tissues were harvested for immunohistological analysis. Lungs were stained with Verhoeff-Van Gieson for the degree of inflammation and muscularization. RV sections were stained with Picrosirius Red for collagen deposition and cardiomyocyte size. Results showed that MR inhibition with SP, at a dose that does not impact systemic blood pressure, significantly reduces RV systolic pressure. However, the attenuation in right ventricular systolic pressure with SP was not reproduced by deletion of MR in SMC or EC. Similarly, MR inhibition attenuates the increase in RV cardiomyocyte area independent of vascular MR with no effect on RV weight or interstitial fibrosis. RV perivascular fibrosis is significantly decreased by SP and this could be reproduced by specific deletion of MR from ECs. MR inhibition significantly reduces pulmonary arteriolar muscularization, independent of EC-MR or SMC-MR. Finally, the degree of perivascular inflammation in pulmonary vessels is attenuated by MR antagonism and is fully reproduced by SMC-specific MR deletion. In conclusion, these studies demonstrate that, in the Sugen/hypoxia PH model, systemic-MR blockade significantly attenuates the PH phenotype and vascular MR has cell-specific effects with EC-MR contributing to RV perivascular fibrosis and SMC-MR driving pulmonary perivascular inflammation. Finally, the role of MR in pulmonary vascular muscularization and RV cardiomyocyte hypertrophy is likely mediated by MR in other non-vascular cell types including cardiomyocytes and/or inflammatory cells.

Myeloid mineralocorticoid receptor contributes to lung inflammation and vascular remodeling in experimental pulmonary hypertension

Guanming Qi, Rod R. Warburton, Krishna C. Penumatsa, Mary E. Moss, Joshua Man, Qing Lu, Seung K. Kim, Nicholas S. Hill, Iris Z. Jaffe and Ioana R. Preston

Pulmonary, Critical Care and Sleep Division, Molecular Cardiology Research Institute, Tufts Medical Center, Boston, MA, USA

Previous studies have indicated that mineralocorticoid receptor (MR) regulates systemic vascular function and contributes to cardiovascular disease by promoting cell proliferation and fibrosis, but the underlying mechanism remains unclear. We hypothesize that myeloid MR plays a pivotal role in the cross-talk between macrophages (MØs) and pulmonary fibroblasts, promoting pulmonary inflammation-mediated vascular remodeling of pulmonary hypertension (PH). Male myeloid-specific MR knockout (MyMR-KO) mice and their littermate controls (MyMR-intact) were exposed to Sugen5416 (Su)/hypoxia to induce PH or normoxia for four weeks. We measured right ventricular (RV) hemodynamics, cardiomyocyte size, pulmonary vascular muscularization and collagen deposition, and perivascular lung inflammation. In vitro, human pulmonary adventitial fibroblasts (hFibs) were treated with conditioned medium from MyMR-KO MØs (vs. MyMR-intact) and expression of profibrotic genes was measured. Results showed that Su/hypoxia produced similar increases in RV systolic pressure and RV mass in both MyMR-KO and MyMR-intact mice. However, MyMR-KOs showed attenuated pulmonary vascular remodeling compared with MyMR-intact mice with decreased muscularization and arterial wall collagen deposition. RV cardiomyocyte area was also reduced in MyMR-KO mice. Moreover, lung inflammation was attenuated in MyMR-KO mice with down-regulation of TNFα expression and reduced infiltration of Galectin-3 (Gal-3)-positive cells in the lungs compared to MyMR-intact mice. When exposed to conditioned medium from MyMR-KO MØs, cultured hFibs significantly decreased mRNA expression of profibrotic genes: collagen Iα1 (Col IA1), transglutaminase 2, and α-smooth muscle actin, but not Ki67, a proliferative gene. In conclusion, our results suggest that MR in MØs contributes to experimental PH via increased accumulation of Gal-3-positive inflammatory cells in the perivascular area, followed by production of paracrine factors that activate adventitial fibroblasts toward a profibrotic phenotype.

Exhaled nitric oxide is not a biomarker for idiopathic pulmonary arterial hypertension or treatment efficacy

Esmaeil Mortaz, Majid Malekmohammad, Gert Folkerts, Babak S. Kashani, Parisa A. Naghan, Zahra H. Dastenae, Batoutl Khoundabi and Ian M. Adcock

Clinical Tuberculosis and Epidemiology Research Center, Tracheal Disease Research Center, Chronic Respiratory Diseases Research Center, National Research Institute of Tuberculosis and Lung Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran; Division of Pharmacology, Utrecht University, Utrecht, Netherlands; Helal-e-Iran Applied Science Higher Education Institute (Red Crescents Society of Iran), Tehran, Iran; Cell and Molecular Biology Group, National Heart and Lung Institute, Imperial College London, London, UK; Priority Research Centre for Asthma and Respiratory Disease, Hunter Medical Research Institute, University of Newcastle, Newcastle, NSW, Australia

Idiopathic pulmonary arterial hypertension (IPAH) is a fatal illness. Despite many improvements in the treatment of these patients, there is no unique prognostic variable available to track these patients. The aim of this study was to evaluate the association between fractional exhaled nitric oxide (FeNO) levels, as a noninvasive biomarker, with disease severity and treatment outcome. A total of 36 patients (29 women and 7 men, mean age 38.4 ± 11.3 years) with IPAH referred to the outpatient’s clinic of Masih Daneshvari Hospital, Tehran, Iran, were enrolled into this pilot observational study. Echocardiography, six-minute walking test (6MWT), FeNO, brain natriuretic peptide (BNP) levels, and the functional class of patients were assessed before patients started treatment. Assessments were repeated after three months. Thirty healthy non-IPAH subjects were recruited as control subjects. Results showed that there was no significant difference in FeNO levels at baseline between patients with IPAH and subjects in the control group. There was no significant increase in FeNO levels during the three months of treatment and levels did not correlate with other disease measures. In contrast, other markers of disease severity were correlated with treatment effect over the three months. In conclusion, FeNO levels are a poor non-invasive measure of IPAH severity or of treatment response in these patients in this pilot study.

Genetic predisposition to high altitude pulmonary edema

Christina A. Eichstaedt, Heimo Mairbäurl, Jie Song, Nicola Benjamin, Christine Fischer, Christoph Dehnert, Kai Schommer, Marc M. Berger, Peter Bärtsch, Ekkehard Grünig and Katrin Hinderhofer

Centre for Pulmonary Hypertension, Thoraxclinic at the University Hospital Heidelberg, Heidelberg, Germany; Laboratory of Molecular Genetic Diagnostics, Institute of Human Genetics, Heidelberg University, Heidelberg, Germany; Translational Lung Research Center Heidelberg, Heidelberg, Germany; Center for Lung Research (DZL), Heidelberg, Germany; Department of Sports Medicine, Medical Clinic VII, University Hospital Heidelberg, Heidelberg, Germany; Medbase Checkup Center, Zurich, Switzerland; Department of Cardiovascular Medicine, The Second Xiangya Hospital, Central South University, Changsha, China; Department of Anesthesiology, Perioperative and General Critical Care Medicine, University Hospital Salzburg, Paracelsus Medical University, Salzburg, Austria

Exaggerated pulmonary arterial hypertension (PAH) is a hallmark of high-altitude pulmonary edema (HAPE). The objective of this study was therefore to investigate genetic predisposition to HAPE by analyzing PAH candidate genes in a HAPE-susceptible family and in unrelated HAPE-susceptible mountaineers. Eight family members and 64 mountaineers were clinically and genetically assessed using a PAH-specific gene panel for 42 genes by next-generation sequencing. Results showed that two otherwise healthy family members, who developed re-entry HAPE at 3640 m during childhood, carried a likely pathogenic missense mutation (c.1198T > G p.Cys400Gly) in the Janus Kinase 2 gene. One of them progressed to a mild form of PAH at the age of 23 years. In 2 of the 64 HAPE-susceptible mountaineers, likely pathogenic variants have been detected, one missense mutation in the Cytochrome P1B1 gene and a deletion in the Histidine-Rich Glycoprotein gene. In conclusion, this is the first study identifying an inherited missense mutation of a gene related to PAH in a family with re-entry HAPE showing a progression to borderline PAH in the index patient. Likely pathogenic variants in 3.1% of HAPE-susceptible mountaineers suggest a genetic predisposition in some individuals that might be linked to PAH signaling pathways.

Chronic thromboembolic pulmonary hypertension—genetic findings

Christina A. Eichstaedt, Jeremias Verweyen, Nicola Benjamin, Christine Fischer, Michael Halank, Eckhard Mayer, Stefan Guth, Christoph Wiedenroth, Jie Song, Benjamin Egenlauf, Satenik Harutyunova, Panagiota Xanthouli, Alberto M. Marra, Ralf Ewert, Katrin Hinderhofer and Ekkehard Grünig

Centre for Pulmonary Hypertension, Thoraxclinic at the University Hospital Heidelberg, Heidelberg, Germany; Laboratory of Molecular Genetic Diagnostics, Institute of Human Genetics, Heidelberg University, Heidelberg, Germany; Department of Internal Medicine I, Carl Gustav Carus University Hospital, Technical University of Dresden, Dresden, Germany; Department of Thoracic Surgery, Kerckhoff Clinic, Bad Nauheim, Germany; Department of Internal Medicine B – Cardiology, Intensive Care, Pulmonary Medicine and Infectious Diseases, University of Greifswald, Greifswald, Germany

Possible underlying genetic predisposition to chronic thromboembolic pulmonary hypertension (CTEPH) is largely unexplored. Approximately 85% of hereditary pulmonary arterial hypertension (PAH) patients carry pathogenic variants within the bone morphogenic protein receptor 2 (BMPR2) gene or associated pathway genes. Only few case reports of CTEPH patients with pathogenic variants exist. Hence, genetic diagnostics is not recommended by current guidelines for this patient population. Thus, the objective of this study was to analyze the prevalence of pathogenic variants in PAH genes in CTEPH patients. A systematic screening was conducted for pathogenic variants in 13 PAH and 29 additional candidate genes using a PAH-specific gene panel based on next-generation sequencing. CTEPH was clinically diagnosed according to current guidelines. Results showed that out of 40 CTEPH patients, four (10%) carried pathogenic variants, eight further patients (20%) carried variants of unknown significance in three PAH and seven candidate genes. One patient had a nonsense mutation (c.2071A > T p.Lys691*) in the BMPR2 gene and three further patients carried a pathogenic variant (missense mutation, c.1849G > T p.Val617Phe) in the Janus Kinase 2 (JAK2) gene, which led to a myeloproliferative disease in each variant carrier. The prevalence of this JAK2 variant was significantly higher than expected (p < 0.0001). In conclusion, CTEPH patients may have a genetic predisposition more often than previously thought. The predisposition for myeloproliferative diseases could be an additional risk factor for CTEPH development. Thus, genetic diagnostics may be considered also for CTEPH patients.

Genetic findings in patients with different forms of pulmonary hypertension

Christina A. Eichstaedt, Zoe Saßmannshausen, Henning Gall, Hans-Jürgen Seyfahrt, Marianne Lerche, Michael Halank, Panagiota Xanthouli, Satenik Harutyunova, Benjamin Egenlauf, Katrin Milger-Kneidinger, Stefan Rosenkranz, Ralf Ewert, Mareike Lankeit, Tobias Lange, Katrin Hinderhofer and Ekkehard Grünig

Center for Pulmonary Hypertension, Thoraxklinik at Heidelberg University Hospital, Translational Lung Research Center Heidelberg, German Center for Lung Research, Heidelberg, Germany; Laboratory for Molecular Diagnostics, Institute of Human Genetics, Heidelberg University, Heidelberg, Germany; Department of Pneumology, Medical and Policlinic II, University Hospital of Gießen and Marburg, Universities of Giessen and Marburg Lung Center, Gießen, Germany; Department of Pneumology, Medical Clinic II, University Hospital of Leipzig, Leipzig, Germany; Medical Clinic I, University Hospital of Dresden, Dresden, Germany; Department of Internal Medicine V, Ludwig-Maximilian University of Munich, Munich, Germany; Asklepios Clinic Gauting, Comprehensive Pneumology Centre Munich, German Center for Lung Research, Munich, Germany; Department III of Internal Medicine and Cologne Cardiovascular Research Center, Cologne University Heart Center, Cologne, Germany; Department of Internal Medicine B – Cardiology, Intensive Care, Pulmonary Medicine and Infectious Diseases, University of Greifswald, Greifswald, Germany; Department of Internal Medicine and Cardiology, Campus Virchow-Klinikum, Charité – University Medicine, Berlin, Germany; Division of Pneumology, Department of Internal Medicine II, University Medical Center Regensburg, Regensburg, Germany

A genetic predisposition is known for different forms of pulmonary arterial hypertension (PAH). Most pathogenic variants were identified in bone morphogenetic protein receptor type 2 (BMPR2) in hereditary PAH. However, further PAH genes have been described. The aim of this study was to screen for all known PAH genes in a large cohort of patients with PAH and other forms of pulmonary hypertension (PH) who have been referred for genetic testing. DNA from 244 patients was extracted and sequenced using a PAH-specific gene diagnostics panel for: ACVRL1, BMPR1B, BMPR2, CAV1, EIF2AK4, ENG, GDF2, KCNA5, KCNK3, KLF2, SMAD4, SMAD9, and TBX4. Of these, 180 samples were also sequenced for ATP13A3, AQP1, and SOX17. Results showed that of the 244 patients, 48% were classified as idiopathic pulmonary arterial hypertension (IPAH), 17% as hereditary pulmonary arterial hypertension (HPAH), 11% as congenital heart disease-associated PAH, 6% as connective tissue disease-associated PAH, and 8% as pulmonary veno-occlusive disease (PVOD). Included were also patients with sarcoidosis-associated PH (n = 7), Osler–Weber–Rendu syndrome (HHT, n = 6), portal hypertension (n = 3), persistent PH of the newborn (n = 3), and drug-induced PAH (n = 3). Of the 60 pathogenic variants identified, 53% were located in the BMPR2 gene. The remaining variants were found in the genes ACVRL1, AQP1, ATP13A3, EIF2AK4, ENG, GDF2, KCNA5, KCNK3, SMAD9, SOX17, and TBX4. HPAH patients carried pathogenic variants in BMPR2 but also in AQP1, EIF2AK4, KCNK3, SMAD9, and SOX17. Patients with PVOD and HHT showed pathogenic variants in EIF2AK4, ENG, and ACVRL1. Two sarcoidosis-associated PH patients carried pathogenic variants in the genes GDF2 and SOX17. In conclusion, pathogenic variants were located in BMPR2 and in further PAH genes. Genetic predisposition was not restricted to HPAH, IPAH, and PVOD but could also be identified in PH with sarcoidosis and other forms of associated PAH. Thus, genetic diagnostics might be useful in a larger patient cohort than currently recommended in the guidelines.

Hemodynamic phenotypes in patients with systemic sclerosis screened for pulmonary hypertension (PH)—the impact of the new definition of PH

Panagiota Xanthouli^*, Suzan Jordan^*, Nicklas Milde, Alberto M. Marra, Christina Eichstaedt, Norbert Blank, Benjamin Egenlauf, Mathias Gorenflo, Satenik Harutyunova, Hanns-Martin Lorenz, Christian Nagel, Vivienne Theobald, Silvia Ulrich, Ekkehard Grünig, Nicola Benjamin and Oliver Distler^*

Centre for Pulmonary Hypertension, Thoraxklinik at Heidelberg University Hospital, Heidelberg, Germany; Translational Lung Research Center Heidelberg, Member of the German Center for Lung Research, Heidelberg, Germany; Department of Rheumatology, University Hospital Zurich, Zurich, Switzerland; IRCCS SDN, Naples, Italy; Laboratory for Molecular Diagnostics, Institute of Human Genetics, Heidelberg University, Heidelberg, Germany; Department of Internal Medicine V: Hematology, Oncology and Rheumatology, University Hospital Heidelberg, Heidelberg, Germany; Department of Pediatric Cardiology, University Hospital Heidelberg, Heidelberg, Germany; Lung Centre, Klinikum Mittelbaden, Baden-Baden Balg, Baden-Baden, Germany; Department of Pulmonology, University Hospital Zurich, Zurich, Switzerland

^*These authors contributed equally.

In this study, we investigated the impact of the new hemodynamic definition of pulmonary hypertension (PH) with mean pulmonary arterial pressure (mPAP) > 20 mmHg, pulmonary arterial wedge pressure ≤15 mmHg, and pulmonary vascular resistance (PVR) ≥3 WU, as proposed by the 6th PH World Symposium in patients with systemic sclerosis (SSc). In consecutive SSc patients who were prospectively screened for PH, including right heart catheterization in two PAH/SSc centers (Heidelberg and Zurich), hemodynamic and clinical variables have been reassessed according to the new PAH definition. Long-term follow-up and survival analysis have been performed. Patients with significant lung or left heart disease were excluded from comparative analyses. Results showed that the final dataset included 284 SSc patients who have been followed for 3.7 ± 3.7 (median 3.4) years, 146 patients (49.2%) had mPAP ≤20 mmHg, 19.3% had mildly elevated mPAP (21–24 mmHg), and 29.4% had mPAP ≥25 mmHg. In the group of mildly elevated mPAP, only four patients (1.4% of the whole SSc cohort) had PVR values ≥3 WU and could be reclassified as manifest SSc-Associated pulmonary arterial hypertension (APAH) according to the new definition. If a threshold for PVR ≥2 WU would be used, 28 (9.85%) patients would have been newly diagnosed with PAH. These patients (mPAP 21–24 mmHg, PVR ≥2 WU) had already an early pulmonary vascular disease with reduced six-minute walking distance (p < 0.001), tricuspid annular plane systolic excursion (p = 0.004), pulmonary arterial compliance (p < 0.001), and long-term survival (p = 0.002) compared to those with normal hemodynamic values. In conclusion, the new hemodynamic PAH definition did not have a significant impact on the diagnosis of PH. The data of this study suggest that a PVR threshold > 3 WU may be too high and significantly impairs the possibility of an early diagnosis with significant impact on survival.

Testing of severe pulmonary hypertension with sildenafil in heart transplant candidates

Hikmet Al-Hiti and Vojtech Melenovsky

Department of Cardiology, IKEM, Prague, Czech Republic

Some patients with advanced heart failure (HF) develop severe pulmonary hypertension (PH). Testing of PH reversibility is therefore used for selection of transplantation (Tx) candidates. The aim of our study was to determine the acute effect of i.v. sildenafil to affect pulmonary hypertension and safely administer it. We performed 538 right heart catheterization (RHC) (from 2017 to 2018) as a part of pre-Tx work-up. In 26 patients (4.8%), pulmonary hemodynamics mandated testing of reversibility for relevant precapillary component of PH (transpulmonary pressure gradient (TPG) > 15 mmHg or pulmonary vascular resistance (PVR) >3 WU in euvolemia). RHC was performed using thermodilution SG catheter. Hemodynamic parameters were measured at the baseline and after 10 min of single dose i.v. of sildenafil 20 mg (Revatio, Pfizer). Drug-induced changes from baseline were compared with paired t statistics. Results are summarized in Table 1. Sildenafil reduces PVR and Pulmonary capillary wedge pressure (PCWP) and increases cardiac output (CO).

In conclusion, prevalence of precapillary PH in current HF pre-heart transplantation (HTx) population is lower than reported previously. Testing the reversibility of PH in heart transplant candidates, i.v. sildenafil is safe and appears to be a very good alternative for minimal effect on systemic resistance. We observed no untoward effects of Phosphodiesterase-5 inhibitors (PDE5i), such as systemic hypotension or worsening pulmonary congestion with Pulmonary Artery Wedge Pressure (PAWP) increase.

Table 1.

▪

	Rest	Sildenafil	Delta
Heart rate, /min	79 ± 18.7	77 ± 18.3	–1.5
PA mean, mmHg	42 ± 7.4	30.4 ± 8.5	–11.6
PA wedged pressure, mmHg	25 ± 5.7	19 ± 7.1	–6
Cardiac output, l/min	3.6 ± 0.9	4.2 ± 1	0.65
Pulmonary vascular resistance, WU	4.9 ± 1.6	2.8 ± 5.8	–2.09
Total systemic resistance, WU	29 ± 9.6	15.2 ± 8.2	–7.85
Total systemic resistance (TSR)/PVR	4.7 ± 1.9	7.0 ± 13.1	2.29

PA, pulmonary artery; PVR, pulmonary vascular resistance.

Pulmonary arterial hypertension registration in Iran (“10 years” experience)

Majid Malekmohammad, Esmaeil Mortaz, Babak S. Kashani and Parisa A. Naghan

Tracheal Disease Research Center, Clinical Tuberculosis and Epidemiology Research Center, Chronic Respiratory Diseases Research Center, National Research Institute of Tuberculosis and Lung Diseases (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran

Pulmonary hypertension (PH) is a hemodynamic and pathophysiological condition defined as increase in mean pulmonary arterial pressure ≥25 mmHg at rest as assessed by right heart catheterization. It can be due to a primary elevation of pressure in the pulmonary arterial system alone (pulmonary arterial hypertension) or secondary to elevations of pressure in the pulmonary venous and capillary systems (pulmonary venous hypertension). Pulmonary arterial hypertension (PAH) can be idiopathic (IPAH), hereditable (HPH), or associated with other conditions such as connective tissue disease, congenital heart disease, portal hypertension, human immunodeficiency virus infection, anorexigen exposure, or schistosomiasis. Not much information about the PAH from Iranian population is available, thus in this study, we investigate the PH patients’ demographics and etiology information in referred patient in Hospital for 10 years. This registration profiles were designed based on guidelines of ERS/ESC and ISHLT. For this registration, all information of IPAH and PAH patients was applied to the website of Iranian pulmonary hypertension and follows-up the patient’s demographic information. A total of 353 PH patients (225 Female and 125 Male) referred to Masih Daneshvari Hospital, Tehran, Iran, between 2009 and 2019 were studied. Among them, 182 patients were characterized as IPAH (n = 121 F and n = 61 M). The average age of PH patients was 48.6 ± 16 years for the IPAH and 48.2 ± 15 years for primary PH. The etiology of most primary PH patients was Eisenmenger Syndrome (43 patients, 12.2%), collagen vascular (22 patients, 6.2%), and chronic thromboembolic PH (95 patients, 26.9%). Evaluation of functional class based on New York Heart Association shows 15 patients with FC I (4.6%), 177 patients with FC II (50.7%), 142 patients with FC III (40.7%), and 14 patients with FC IV (4%). In treatment strategy, 209 patients were treated with Tadalafil, 230 patients with Bosentan, and 65 patients with ILomedin. Due to the economy sanction in the land, we have limited access to standard advance PH drugs for treatment. In conclusion, diagnosis, management, and follow-up of PH, and certainly IPAH patients under Iranian PAH registry system (website www.ipah.ir) during 10 years, leads to organizing the patient care system and management and also causes to obtain the preclinical data.

Inhaled AAV1.hSIN3a gene therapy restores BMPR2 expression and attenuates pulmonary arterial hypertension

Malik Bisserier, Firas Elmastour, Shihong Zhang, Peter Dorfmüller, Marc Humbert, Yassine Sassi, Thomas Weber, Roger J. Hajjar and Lahouaria Hadri

Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York, NY, USA; Translational Medicine Group, Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, MA, USA; Hospital Antoine-Béclère, Clamart, France; INSERM U999, Centre Chirurgical Marie-Lannelongue, Le Plessis-Robinson, France; Phospholamban Foundation, Amsterdam, Netherlands

Pulmonary arterial hypertension (PAH) is a rare and fatal cardiopulmonary disease characterized by an elevation of pulmonary artery pressure (PAP) and vascular resistance, which can lead to right ventricular (RV) failure and ultimately death. Despite the rapid accumulation of data over the past decade, a complete understanding of the molecular mechanisms in PAH is still lacking. Therefore, the identification and characterization of new therapeutic targets remain critical. Several studies have previously suggested that hypermethylation of the Bone Morphogenetic Protein Receptor Type 2 (BMPR2) promoter may be associated with BMPR2 downregulation. Here, we investigated for the first time the role of Switch-Independent 3a (SIN3a) in the epigenetic regulation of BMPR2 in PAH. We used lung samples from PAH patients, preclinical rodent PH models, and human pulmonary artery smooth muscle cells (hPASMC). The expression of SIN3a was modulated using a lentivirus vector/siRNA in vitro and an adeno-associated virus encoding human SIN3a (AAV1.hSIN3a) in vivo. Our results revealed a significant decrease of SIN3a expression in lung samples from PAH patients and PH animal models. In vitro, we found that SIN3a overexpression inhibits hPASMC proliferation, migration, and upregulates BMPR2 expression. SIN3a knockdown showed opposite effects. Using mRNA sequencing, we found that SIN3a repressed the gene expression of several methyltransferases while promoting the expression of demethyltransferases. Mechanistically, we found that SIN3a restored BMPR2 expression by decreasing the BMPR2 promoter methylation. Finally, using experimental PH rodent models, our result showed that intratracheal administration of AAV1.SIN3a significantly decreased Monocrotaline and Sugen/Hypoxia-induced PAH. AAV1.hSIN3a gene therapy significantly inhibited pulmonary vascular and RV remodeling, decreased PAP and RV pressure, and successfully restored BMPR2 expression. In Conclusion, our study uncovered for the first time the role of SIN3a in PH and identified a novel molecular mechanism involved in the methylation of the BMPR2 promoter. This study identified SIN3a gene therapy as a new promising therapeutic strategy for treating PAH patients.

The impact of LOXL2 inhibition on pulmonary arterial hypertension

Jochen Steppan, Huilei Wang, Sandeep Jandu, Kavitha Nandakumar, Steven S. An, Dan E. Berkowitz, Larissa Shimoda and Lakshmi Santhanam

Departments of Medicine, Anesthesiology, Johns Hopkins University, Baltimore, MD, USA

Conduit pulmonary arterial stiffening and the resultant increase in pulmonary vascular impedance has emerged as an important underlying driver of pulmonary arterial hypertension (PAH) contributing to right heart failure. Thus, anti-remodeling therapy could be beneficial in decelerating disease progress. Given that matrix deposition is central to vascular remodeling, we evaluated the role of the collagen crosslinking enzyme lysyl-oxidase-like-2 (LOXL2) in this study. Human pulmonary artery (PA) endothelial cells subjected to hypoxia showed increased LOXL2 secretion into the media, while LOXL2 abundance in the cytosol is decreased. Rats were injected with either SU5416 followed by continuous exposure to hypoxia (SuHx) or exposed to monocrotaline (MCT) and placed at room air for four weeks, with half of the animals treated with PAT1251 (LOXL2 inhibitor). Animals in the PAH groups had markedly increased Fulton indices, while animals treated with PAT1251 were protected. The stress–strain relationship, indicative of passive vascular stiffness, shifted upward in SuHx and MCT rats while fully recovered with LOXL2 inhibition. Vascular reactivity of isolated PAs showed decreased contractility and impaired endothelial function in PAH animals that was absent in controls. Right ventricular pressure–volume loops demonstrate restored PA pressures and arterial elastance. Furthermore, right heart catheterization data were also corroborated in a LOXL2^+/– mouse model, which we showed increased PA pressures and arterial elastance in wildtypes subjected to SuHx, while LOXL2^+/– animals were protected. In conclusion, at a cellular and tissue level, hypoxia results in increased LOXL2 secretion. This coincides with increased endothelial dysfunction, decreased smooth muscle cell contractility, and increased vessel stiffness. Animals exposed to SuHx or MCT demonstrate higher PA pressures, increased right ventricular elastance, and impaired cardiac relaxation. Pharmacological LOXL2 inhibition or genetic knockdown is protective in models of PAH. This data suggest evaluating LOXL2’s role in pulmonary hypertension in more detail.

Experimental validation and refinement of a mathematical model of profibrotic cell signaling in pulmonary arterial adventitial fibroblasts

Ariel Wang and Daniela Valdez-Jasso

Department of Bioengineering, University of California, San Diego, CA, USA

Pulmonary arterial adventitial fibroblasts (PAAFs) are important regulators of fibrotic vascular remodeling during pulmonary arterial hypertension (PAH). PAAFs can be activated to exhibit different functional characteristics that contribute significantly to pulmonary vascular remodeling and stiffening. However, currently no therapies have been found to reverse matrix remodeling and vascular stiffening that occurs during PAH. In order to better identify treatments to target adventitial fibrosis, we sought to investigate the interplay between mayor pathways that have been reported to regulate myofibroblast transformation and extracellular matrix expression in PAAFs. Here, we describe an initial validation of a mathematical model with published experimental data. Based on a thorough literature review, we built a PAAF signaling network model using 53 papers that include observed effects of mechanical stimulation and seven main pathways: Hippo, phosphoinositide-3-kinase, transforming growth factor β, Notch, reactive oxygen species, angiotensin II, and calcineurin-NFAT. Model outputs include expression of matrix proteins and lysyl oxidase, fibroblast proliferation, migration, and myofibroblast transformation. We implemented the model as a predictive computational model to investigate combinatorial inhibition of critical pathways affected by fibrosis. Results show that the model was validated using 21 independent papers of rat and human PAAF data and were able to correctly predict 80% input–output experimental results. Without optimizing parameters, the model also accurately captured the trends reported in independent dynamic inhibition experiments carried out by Zhang and He et al. of isolated rat PAAFs. In conclusion, we expect that optimizing parameters will make the model accurate enough to generate new hypotheses and design key experiments to investigate the potential combinatorial therapeutics to reverse vascular remodeling.

Haematopoiesis—a role for bone morphogenetic protein type 2 receptor?

Alexi Crosby, Michael Newnham, Charaka Hadinnapola, Emily Groves, Emilia M. Swietlik, Stephen Moore, Mark Toshner and Nicholas W. Morrell

UK National Institute for Health Research BioResource Rare Diseases Consortium, UK Pulmonary Arterial Hypertension Cohort Study Consortium, Department of Clinical Medicine, University of Cambridge, Cambridge, UK

Increasing evidence suggests a link between abnormalities in the myeloid cell lineage and pulmonary arterial hypertension (PAH). Heterozygous mutations in the gene encoding the bone morphogenetic protein type 2 receptor (BMPR2) are the most common genetic cause of PAH. We sought to characterise the impact of the genetic loss of BMPR2 in the myeloid lineage in humans and mice. In a large cohort of PAH patients, with (n = 118) and without (n = 570) BMPR2 mutations, chronic thromboembolic pulmonary hypertension (CTEPH) patients (368) and controls (2977), blood count indices were analysed. Mx1-cre mice were crossed with bmpr2^flox/flox mice. Cre-recombinase was induced with polyinosinic–polycytidylic acid (Poly I:C). Twelve weeks post-induction, mice underwent right-heart catheterisation, exsanguination and tissue removal. Results show that 12 weeks after induction of cre-recombinase, we observed significant increases in red blood cells, haematocrit and haemoglobin in Mx1-cre/bmpr2^flox/flox mice compared with bmpr2^flox/flox mice (p < 0.05). A significant increase in circulating monocytes, granulocytes as well as a significant increase in bone marrow haematopoeitic stem cells, monocytes and granulocytes and megakaryocytes in the femurs (p < 0.05) were also observed. PAH patients with a BMPR2 mutation had significant increases (p < 0.05) in haemoglobin, haematocrit and white blood cells. These differences were also seen in PAH patients, with and without BMPR2 mutations, compared with controls and CTEPH patients. In conclusion, we have identified a role for bmpr2 in the differentiation of the human and mouse haematopoietic system. Similarities between PAH patients with a mutation in BMPR2 and mice with bmpr2 knocked out of the myeloid lineage were observed. Interestingly PAH patients with and without a BMPR2 mutation were phenotypically more similar to each other than either control group (controls/CTEPH), thus indicating the importance of BMPR2 but not its exclusivity. More work is required to determine the exact contribution of BMPR2-deficient bone marrow to the development of PAH.

Implication of the histone methyltransferase “G9a” in pulmonary arterial hypertension

Awada Charifa, Habbout Karima, Nadeau Valérie, Breuils-Bonnet Sandra, Paulin Roxane, Provencher Steeve, Boucherat Olivier and Bonnet Sébastien

Pulmonary Hypertension Research Group, Centre de Recherche de l’Institut Universitaire de Cardiologie et de Pneumologie de Québec, Québec, QC, Canada

Pulmonary arterial hypertension (PAH) is a cardiopulmonary disorder characterized by persistent elevation of pulmonary arterial (PA) pressure and premature death. PA smooth muscle cells (PASMCs) from PAH patients present a cancer-like hyperproliferative and apoptosis-resistant phenotype contributing to vasoconstriction and remodeling of distal PAs. Although epigenetic alterations are increasingly appreciated as a contributing factor of PAH development, one important challenge in epigenetic therapy is defining which genes are the drivers. G9a is an epigenetic factor, known to be overexpressed in many cancers, promoting cell proliferation and survival. Given that PAH and cancer cells share numerous similarities, it is crucial to determine if G9a is implicated in PAH. We thus hypothesized that the inhibition of G9a could reduce the pro-proliferative and apoptosis-resistance phenotype of PAH-PASMCs. Using western blot (WB) and immunofluorescence, we demonstrated that G9a is significantly overexpressed in distal PAs and isolated PASMCs from PAH patients (n = 6–14, p < 0.01). Similarly, G9a was increased (WB and immunofluorescence, p < 0.05) in two models mimicking the disease; namely the monocrotaline rat model and mice exposed to chronic hypoxia. In vitro, we found that pharmacological inhibition of G9a using BIX01294 and UNC0642 dose-dependently reduces PAH-PASMC proliferation (Ki67 labeling and EdU assay, p < 0.001) and survival (Annexin V assay, p < 0.001). Inhibition of G9a was accompanied by the appearance of numerous cytoplasmic vacuoles. As detected by WB and immunofluorescence, inhibition of G9a in PAH-PASMCs resulted in a significant increase in LC3-II accumulation which was in parallel with an increase in p62; thus, suggesting that inhibition of G9a interferes with autophagic flux. In conclusion, we showed for the first time that G9a is overexpressed in human and experimental PAH contributing to the pro-proliferative and anti-apoptotic phenotype of PAH-PASMCs. Further research is required to understand precisely the role of G9a in the abnormal phenotype of PAH-PASMCs.

Integrative single-cell omics to uncover mechanistic insights and drug repurposing candidates for pulmonary arterial hypertension

Jason Hong, Douglas Arneson, Soban Umar, Gregoire Ruffenach, In S. Ahn, Graciel Diamente, May Bhetraratana, Christine Cunningham, Mansoureh Eghbali and Xia Yang

Division of Pulmonary & Critical Care, Departments of Medicine, Integrative Biology & Physiology, Anesthesiology, University of California, Los Angeles, CA, USA

Despite many years of translational research efforts, pulmonary arterial hypertension (PAH) remains an incurable and devastating disease for which novel approaches to drug discovery are critically needed. In a systematic and unbiased approach, we sought to leverage single-cell RNA sequencing (scRNA-seq) with transcriptional signatures of drugs to predict existing drugs to reposition for PAH. Lung scRNA-seq was performed on Sugen/hypoxia (SuHx), monocrotaline, and control rats (n = 6/group). Cell type-specific PAH gene signatures were used to screen small molecule compounds using Connectivity Map, a genome-scale library of cellular signatures that catalogs transcriptional responses to various compounds. Results show that single-cell analysis of 18 rat lungs yielded 32,899 cells and 28 distinct cell types. TNF-α/NFkB signaling was the most highly upregulated pathway across multiple cell types in both disease models and in independently curated sets of known human PAH genes from DisGeNET and Comparative Toxicogenomics Database. Gene alterations in non-classical monocytes from SuHx showed the strongest upregulation of this pathway and for known human PAH genes. Screening of 2429 compounds for in silico reversal of the non-classical monocyte transcriptional signature revealed the top drug to be treprostinil, currently the most effective PAH-targeted therapy available for patients and thus serving as a form of validation of our approach. Experiments are currently underway to validate other top drugs predicted from this analysis. In conclusion, our scRNA-seq study offers comprehensive insights into the PAH cellular landscape at single-cell resolution, and TNF-α/NFkB signaling and non-classical monocytes emerged as particularly important molecular and cellular targets of PAH. By combining cell type-specific gene disruptions with in silico high-throughput drug screening, we identified candidate drugs for repurposing in PAH.

Inflammatory biomarker profiles at baseline and one-year follow-up in pulmonary arterial hypertension (PAH) and chronic thromboembolic pulmonary hypertension (CTEPH)

T. Koudstaal, Hulst J.A.C. van, I.M. Bergen, P. Heukels, L.W. Geenen, V.J.M. Baggen, den Bosch A.E. van, den Toorn L.M. van, P.P. Chandoesing, J.G.J.V. Aerts, M. Kool and K.A. Boomars

Departments of Pulmonary Medicine, Cardiology, Erasmus MC, Rotterdam, The Netherlands

Pulmonary hypertension (PH) is a devastating disease characterized by increased pulmonary arterial pressure, leading to right heart failure and death. Accumulating evidence is showing increased levels of circulating cytokines, associated with poor prognosis, in pulmonary arterial hypertension (PAH) and chronic thromboembolic pulmonary hypertension (CTEPH) patients. Currently, it is unknown if these circulating cytokine levels are already elevated at diagnosis, and if different cytokine profiles exist in different PAH subsets. Our aim was to investigate circulating inflammatory biomarkers at diagnosis (treatment-naïve/baseline) and after one-year follow-up in PAH-subsets and CTEPH patients. Plasma samples from 50 PAH patients (16 idiopathic (I)PAH, 24 Connective Tissue Disease (CTD)-PAH, and 10 Congenital Heart Disease (CHD)-PAH), 37 CTEPH patients, and 18 healthy controls (HC) were measured at diagnosis (baseline) and in a subset of patients at one-year follow-up for circulating inflammatory biomarkers (VEGF-A, TGF-β, CXCL-9, CXCL-13, interleukin (IL)-1β, IL-6, IL-8, IL-10) and were related to hemodynamic parameters. Results show that at baseline, plasma IL-6 concentrations were elevated in CTD-PAH, IL-10 was increased in IPAH, and TGF-β was significantly enhanced in IPAH and CHD-PAH patients compared to HC. CXCL-9 was elevated in patients (IPAH, CTD-PAH, and CTEPH) compared to HC, whereas CXCL-13 was higher in CTD-PAH compared to controls and CTEPH patients. No differences were observed for VEGF-A, IL-1b, and IL-8. In our cohort, no significant hemodynamic correlations were found between circulating cytokines and hemodynamic parameters at baseline. After one-year follow-up, the concentrations of IL-8, IL-10, and TGF-β increased significantly in paired samples from CTD-PAH patients. In conclusion, our findings indicate that different inflammatory profiles in PH exist at diagnosis, seemingly independent of WHO subgroup classification within PAH subsets and CTEPH. After one-year of treatment, these inflammatory profiles differentially changed for each PH subgroup. Additional analyses are required to determine the prognostic and clinical implications of these results.

Psychosocial and financial burden of medical treatment in pulmonary artery hypertension

Charles D. Burger, Charitha Vadlamudi, Haytham Helmi and Divya P. Menon

Division of Pulmonology, Mayo Clinic, Jacksonville, FL, USA

Treatment of pulmonary arterial hypertension (PAH) has advanced considerably decades with the advent of newer therapeutic medications and the associated improvement in five-year survival. The annual pharmaceutical cost of medical therapy may range from $25,000 to $250,000 and is driven by newer agents and the proven efficacy of combination therapy. The disease and treatment burden for PAH patients are likely intertwined; therefore, we aimed to assess the psychosocial and healthcare implications of PAH therapy on patients. The study was approved by the Mayo Clinic Institutional Review Board. Data were collected prospectively by questionnaire in patients with diagnostic group 1 PAH from two separate cohorts. Cohort 1 was comprised of consecutive patients from Pulmonary Hypertension Clinic at Mayo Clinic Florida (MCF). Cohort 2 was comprised of PAH patients who volunteered for research at 2018 Pulmonary Hypertension Association (PHA) International PH conference (Orlando, FL). A 25-point questionnaire included demographic data, comorbidities, therapy regimens, quality of life, compliance, economic and social challenges of therapy including insurance and out-of-pocket expenses. Patients with group 1 PAH completed the questionnaire after consent. Exclusion criteria included age < 18 years, prior lung transplantation, absence of PAH therapy, and clinical trial participants. Results showed that overall 112 patients, 60 in Cohort 1 (MCF) and 52 in Cohort 2 (PHA), completed the questionnaire. The majority were middle-aged, Caucasian women with either idiopathic (52/112, 46%) or connective tissue disease (28, 25%) associated PAH. Drug therapy spanned all classes with 74% (83/112) on combination therapy. Nonetheless, most patients (91/112, 81%) endorsed active cardiopulmonary symptoms including dissatisfaction with their level of activity despite their current PAH regimen. A minority (12/112, 11%) endorsed the ability to perform two hours of physical work without significant limitations. Participants reported a steady decline in employment rates at the time of diagnosis and with progression of disease with about one-half applying for disability. While most (108/112, 96%) had insurance coverage, out-of-pocket medication expenses were common and most reported that the drug costs were unaffordable without insurance or assistance programs. Statistically, Cohort 1 was slightly older (62 ± 11 vs. 51 ± 14, p < 0.01), had more Caucasians (100% vs. 86%, p = 0.0007), and had different diagnostic classifications (e.g. less idiopathic and heritable PAH, p < 0.001, Table 1), but otherwise there was little difference in treatment, activity level, or psychosocial and financial burden. In conclusion, despite combination PAH therapy in most patients, a significant number of the study population reported that they had persistent cardiopulmonary symptoms, decreased activity levels, steady decline in employment rates, and associated application for disability benefits. Most had insurance coverage, but ongoing out-of-pocket medication expenses were common. Comparison of cohorts from a single center (MCF Cohort 1) to a more demographically heterogeneous group that attended the PHA conference (Cohort 2) did not reveal significant differences in disease or psychosocial and financial burden.

Table 1.

PAH diagnostic classification (p < 0.001).

Diagnostic class	Combined (n = 112)	MCF (n = 60)	PHA (n = 52)
Idiopathic PAH	52	22 (20%)	30 (27%)
Familial PAH	5	0 (0%)	5 (5%)
Portopulmonary hypertension	4	4 (3.6%)	0 (0%)
Adult congenital heart disease-associated PH	11	9 (8%)	2 (2%)
Connective tissue disease-associated PAH	28	20 (18.02%)	8 (7.21%)
Drug-induced PAH	1	0 (0%)	1 (0.9%)
Etiology unknown	8	2	6

MCF, Mayo Clinic Florida; PHA, Pulmonary Hypertension Association; PAH, pulmonary arterial hypertension; PH, pulmonary hypertension.

Endothelial-induced GATA6 deficiency promotes deregulation of Yap/Taz, BMPRII and SOD2 axis, and vascular smooth muscle hyper-proliferation in PAH

Tatiana Kudryashova^*, Asako Ichihara^*, Tetsuo Toyama^*, Yuanjun Shen, Dmitry Goncharov, Theodore Avolio, Robert Lafyatis, Maria Trojanowska^* and Elena Goncharova^*

Department of Medicine, University of Pittsburgh, Pittsburgh, PA, USA; Arthritis Center, Boston University, Boston, MA, USA

^*These first authors contributed equally.

#These senior authors contributed equally.

Increased proliferation and survival of vascular cells in small pulmonary arteries (PA) are key pathological components of pulmonary vascular remodeling in pulmonary arterial hypertension (PAH). Transcriptional factor GATA6 is deficient in PAH pulmonary vasculature, and endothelial-specific Gata6 deficiency induces pulmonary hypertension (PH) in mice. The role and the mechanisms of regulation and function of GATA6 in PA vascular smooth muscle cells (PAVSMC) in PAH remain unstudied. Using single-cell sequencing and immunohistochemical analysis of lung samples and immunoblot analysis of human pulmonary vascular cells from small PAs, we found that GATA6 is significantly downregulated in both, PAH pulmonary artery endothelial cell (PAEC) and PAVSMC compared to controls. Media conditioned by PAH PAECs or non-diseased PAECs with siRNA-induced GATA6 depletion induced GATA6 deficiency and growth of non-diseased PAVSMC. Human PAH PAVSMC had increased levels of pro-proliferative/pro-survival Yap/Taz, deficiency of anti-proliferative SOD2, and increased proliferation compared to controls. Incubation in the media conditioned by non-diseased PAECs with siRNA-induced GATA6 depletion as well as direct transfection with siRNA GATA6 reduced expression of SOD2 and BMPR2, induced Yap/Taz accumulation, and increased growth of non-diseased PAVSMC, demonstrating importance of PAEC-induced GATA6 downregulation in establishing PAH-specific PAVSMC phenotype. Confirming our in vitro findings, mice with endothelial-specific Gata6 knockout showed reduced Gata6 protein levels in both, PAEC and PAVSMC and increased muscularization of small PAs. Dimethyl fumarate (DMF), known activator of antioxidant pathways, restored pulmonary arterial pressure and partially reversed right heart hypertrophy in mice with endothelial-specific Gata6 knockout, decreased proliferation, and induced apoptosis in human PAH PAVSMC without affecting GATA6 and Yap/Taz. In conclusion, together, our data demonstrate that GATA6 deficiency in PAH PAEC promotes downregulation of GATA6 in PAVSMC via paracrine mechanism, causing deregulation of Yap/Taz, SOD2, and BMPRII axis, increasing proliferation and survival. DMF could be considered as a potential treatment option to reduce pulmonary vascular cell proliferation and overall PH.

Effect of exercise training on six-minute walking distance in severe precapillary pulmonary hypertension—results from a European multicenter randomized controlled trial

Ekkehard Grünig^*, Alison MacKenzie^*, Andrew J. Peacock, Christina Eichstaedt, Nicola Benjamin, Silvia Ulrich, Stéphanie Saxer, Maurizio Bussotti, Stefano Ghio, Lina Gumbiene, Eglė Palevičiūtė, Elena Jurevičienė, Antonio Cittadini, Anna A. Stanziola, Alberto M. Marra, Gabor Kovacs, Horst Olschewski, Joan-Albert Barberà, Isabel Blanco, Martin Spruit, Frits Fransen, Anton V. Noordegraaf, Abílio Reis, Mário Santos, Sofia G. Viamonte, Heleen Demeyer, Marion Delcroix, Eduardo Bossone^# and Martin Johnson^#

Centre for Pulmonary Hypertension, Thoraxclinic at the University Hospital, Heidelberg, Germany; Translational Lung Research Center Heidelberg (TLRC), Heidelberg, Germany; German Center for Lung Research (DZL), Heidelberg, Germany; Scottish Pulmonary Vascular Unit, Golden Jubilee National Hospital, Glasgow, UK; Laboratory for Molecular Diagnostics, Institute of Human Genetics, Heidelberg University, Heidelberg, Germany; Centre for Pulmonary Hypertension, University Hospital Zurich, Zurich, Switzerland; Cardiac Rehabilitation Department, IRCCS Maugeri Clinical Scientific Institutes, Milan, Italy; Competence Centre of Pulmonary Hypertension, Vilnius University Hospital Santaros Klinikos, Vilnius, Lithuania; Faculty of Medicine, Vilnius University, Vilnius, Lithuania; Department of Cardiac Surgery, University Hospital Salerno, Salerno, Italy; Department of Cardiology, Antonio Cardarelli Hospital, Naples, Italy; Department of Clinical Medicine and Surgery, Federico II University, Naples, Italy; Department of Cardiovascular Imaging, IRCCS S.D.N., Naples, Italy; Division of Pulmonology, Department of Internal Medicine, Medical University of Graz, Graz, Austria; Ludwig Boltzmann Institute for Lung Vascular Research, Graz, Austria; Hospital Clinic IDIBAPS, University of Barcelona, Barcelona, Spain; Biomedical Research Networking Center on Respiratory Diseases, Madrid, Spain; Department of Research and Education, CIRO, Horn, the Netherlands; Department of Pulmonology, VU University Medical Center Amsterdam, Amsterdam, the Netherlands; Department of Internal Medicine, Hospital Geral de Santo António, Porto, Portugal; Centro de Reabilitação do Norte, Porto, Portugal; Department of Pneumology, University Hospital Leuven, Leuven, Belgium

^*These first authors contributed equally.

^#These senior authors contributed equally.

The benefits of exercise training on exercise capacity and quality of life in pulmonary hypertension (PH) have already been shown in several trials. Results of large multicenter studies are still lacking. A specialized training program for patients with precapillary PH was implemented in 11 centers across 10 European countries. Patients were randomized in a training group or a sedentary control group. Patients of the training group performed a 10–30 days in-hospital training including interval ergometer training, dumbbell training, mental gait training, and respiratory therapy. The training was continued at home until week 15. The primary outcome variable was change in six-minute walking distance (6MWD) between baseline and 15 weeks in the two groups. Secondary outcome parameters included quality of life scores (SF-36 questionnaire) and peak oxygen consumption during cardiopulmonary exercise testing. Out of 129 patients who were enrolled, 116 (58 vs. 58) completed the study. Most participating centers implemented this specialized training program for PH for the first time and managed to adapt it to the local health care system. The primary endpoint mean 6MWD significantly improved in the training group vs. control group by 34.1 ± 8.3 m (p < 0.0001). Also, secondary endpoints significantly improved in the training group. Significant improvements of the training group compared to the control group were found in the quality of life parameter mental health (p = 0.004), WHO functional class (Chi-square p = 0.027), and peak oxygen consumption (control-group corrected improvement 0.9 ± 0.5 mL/min/kg, p = 0.048). In conclusion, this is the first randomized, controlled multicenter study providing evidence of the feasibility of exercise training across 10 European countries. Our study results provide further multicenter evidence of the effect of exercise training programs for PH patients as an add-on therapy. Within this study, a standardized specialized training program with in-hospital start for PH patients in different European countries was successfully established.

Pulmonary hypertension screening in systemic sclerosis (SSc) and idiopathic inflammatory myopathic (IIM) patients. A university hospital experience

Tejada R.A. Gómez, H. Laborde, R. Gómez, Poccard. A. Braillard, N. Mastandrea, V. Giovini, M. Monteverde, V. Barrios, M. Mazzei and Blanco M. Vázquez

Hospital de Clínicas “José de San Martín”, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina

Pulmonary hypertension (PH) prevalence in connective tissue diseases (CTD) highlights the value of regular screening. Clinical, serologic, and comorbidities profiles are described in a series of systemic sclerosis (iSSc) and idiopathic inflammatory myopathic (IIM) patients. Retrospective study from an institutional outpatient sample during years 2008–2018 was performed. Demographic and clinical features were evaluated. Pulmonary function tests, CT scans, and immunoserology tests were performed. We defined comorbilities as interstitial lung disease (ILD), chronic thromboembolic disease, and left heart disease. Inclusion criteria were: patients older than 16 years; ACR/EULAR’13 SSC criteria, and EULAR/ACR’17 criteria for IIM. PH was defined with these transthoracic echocardiography (TTE) features: Doppler pulmonary artery systolic pressure ≥40 mmHg; right ventricular enlargement (VD/VI > 1); and interventricular septum flattening. We evaluated 217 patients with CTD: 129 (SSc) and 88 (IIM). TTE features for PH were fulfilled in 17 (13.2%) SSc patients and 5 (5.7%) IIM patients. All of them showed low tolerance to exercise and frequent dyspnea. WHO functional class was ≥III in all. Five patients received right heart catheterization with medium pulmonary arterial pressure > 45 mmHg (results in Table 1). In conclusion: (1) the screening of PH in SSc is mandatory. A similar approach in IMM should be reevaluated; (2) anticentromere antibodies and U1RNP antibodies were indicative of PH in this CTD series; (3) SSc showed association with ILD, expressing PH Group III; (4) frequent comorbilities in SSC are compatible with multiple pathophysiologic mechanisms of PH; and (5) superposition conditions in CTD were frequent.

Table 1.

▪.

	SSC (n = 17)	IIM (n = 5)
Male/female	1/16	¼
Age (median)	62	42
Serological testing antibodies (n/%)
Antinuclear	16 (94)	5 (100)
Antitopoisomerase	5 (29)	0
Anticentromere (ACA)	9 (53)	0
U1RNP	2 (12)	3 (60)

Dynamic contribution of pericytes to the blood vessel remodeling in pulmonary hypertension

Ly Tu, Jennifer Bordenave, Nihel Berrebeh, Raphaël Thuillet, Amélie Cumont, Benjamin L. Vely, Sophie Nadaud, Laurent Savale, Marc Humbert, Alice Huertas and Christophe Guignabert

INSERM UMR_S 999, Hôpital Marie Lannelongue, Le Plessis-Robinson, France; Faculté de Médecine, Université Paris-Sud, Université Paris-Saclay, Le Kremlin-Bicêtre, France; Service de Pneumologie, AP-HP, Centre de Référence de l’Hypertension Pulmonaire Sévère, Hôpital Bicêtre, Le Kremlin-Bicêtre, France; Institute of Cardiometabolism and Nutrition (ICAN), INSERM UMR_S, Hôpital la Pitié Salpêtrière, Paris, France

Excessive accumulation of resident cells within the pulmonary vascular wall represents the hallmark feature of the remodeling occurring in pulmonary arterial hypertension (PAH). Furthermore, we have previously demonstrated that pulmonary arterioles are excessively covered by pericytes in PAH, but this process is not fully understood. The aim of our study was to investigate the dynamic contribution of pericytes in PAH vascular remodeling. In this study, we performed in situ, in vivo, and in vitro experiments. We isolated primary cultures of human pericytes from controls and PAH lung specimens and then performed functional studies (cell migration, proliferation, and differentiation). In addition, to follow-up pericyte number and fate, a genetic fate mapping approach was used with a NG2CreER^TM;mT/mG transgenic mice in a model of pulmonary arteriole muscularization occurring during chronic hypoxia. We identified phenotypic and functional abnormalities of PAH pericytes in vitro, as they overexpress CXCR7 and TGFβRII and, thereby, display a higher capacity to migrate, proliferate, and differentiate into smooth muscle-like cells than controls. In an in vivo model of chronic hypoxia, we found an early increase in pericyte number in a CXCL12-dependent manner, whereas later, from day 7, activation of the canonical TGF-β signaling pathway induces pericytes to differentiate into smooth muscle-like cells. Our findings reveal a pivotal role of pulmonary pericytes in PAH and identify CXCR7 and TGFβRII as two intrinsic abnormalities in these resident progenitor vascular cells that foster the onset and maintenance of PAH structural changes in blood lung vessels.

Decreased glycolysis as metabolic footprint of endothelial cells in chronic thromboembolic pulmonary hypertension

O. Tura-Ceide^*, Valérie F. Smolders^*, C. Rodríguez, I. Blanco, J. Osorio, L. Piccari, C. Bonjoch, P.H.A. Quax, M. Cascante, V.I. Peinado, M. Castellà and Joan A. Barberà

Department of Pulmonary Medicine, Hospital Clínic – Institut d’Investigacions Biomèdiques August Pi i Sunyer, University of Barcelona, Barcelona, Spain; Faculty of Biology, Department of Biochemistry and Molecular Biology, University of Barcelona, Barcelona, Spain; Department of Vascular Surgery, LUMC, Leiden, The Netherlands; Centro de Investigación Biomédica en Red de Enfermedades Respiratorias, Madrid, Spain; Department of Cardiovascular Surgery, Institut Clínic del Tòrax, Hospital Clínic, University of Barcelona, Barcelona, Spain

^*These authors contributed equally.

Chronic thromboembolic pulmonary hypertension (CTEPH) is caused by non-resolution of thrombi that obstruct main pulmonary arteries and secondary cause vascular remodeling of the pulmonary vasculature. Endothelial cells (EC) are an important component in vascular disease development. We speculated that metabolic dysregulation of EC contributes to cellular changes that promote vascular remodeling in CTEPH. This study used endothelial cells (EC-CTEPH) isolated from specimen extracted at pulmonary endarterectomy. Proliferation and 2D–3D function assays were used to assess angiogenic potential. Expression levels of metabolic enzymes were studied at mRNA and protein level. Glucose consumption and lactate production was measured in the supernatant overlying EC-CTEPH and human pulmonary arterial endothelial (HPAE) using blood gas electrolyte and metabolite (BGEM) cards. EC-CTEPH showed a hyperproliferative phenotype and significantly lower mRNA and protein levels of all glycolytic enzymes analyzed compared to HPAE. Transcript levels of pyruvate dehydrogenase kinase 1 (PDK1) and glutamate dehydrogenase 1 (GLUD1), involved in glutamine metabolism, were also downregulated. Fatty acid and pentose phosphate metabolism did not present significant differences between EC-CTEPH and HPAE. A lower glycolytic flux in EC-CTEPH was observed in terms of glucose consumption and lactate production. Functionally, EC-CTEPH has a reduced angiogenic capacity both in vitro and in vivo. Our results showed that EC in human CTEPH are hyperproliferative and present a reduced angiogenesis and glycolytic metabolism compared to healthy EC. TCA and glutamine metabolism were also downregulated in EC-CTEPH. Other metabolic pathways such as fatty acid oxidation (FAO) and pentose phosphate pathway (PPP) did not show a compensatory upregulation. More studies need to be performed to determine the role of our findings in EC-CTEPH pathogenesis.

The wide spectrum of β-thalassemia intermedia induced pulmonary hypertension: the possible role of specific pulmonary arterial hypertension therapy

Anastasia Anthi, Dimitrios Tsiapras, Panagiotis Karyofyllis, Vassilios Voudris, Apostolos Armaganidis and Stylianos E. Orfanos

Pulmonary Hypertension Clinic and 2nd Department of Critical Care, National & Kapodistrian University of Athens, Attikon Hospital, Athens, Greece; Noninvasive Department of Cardiology, Onassis Cardiac Surgery Center, Athens, Greece; Interventional Department of Cardiology, Onassis Cardiac Surgery Center, Athens, Greece

Pulmonary hypertension (PH) has been reported as a frequent complication in patients with β-thalassaemia intermedia (TI). According to the Fifth World Symposium on PH, PH due to haemolytic anaemias is classified in group 5. The use of pulmonary arterial hypertension (PAH)-specific therapy in these patients is based on data derived from other forms of PH, expert opinion and small series or case reports. We report two patient cases with TI and PH; the first patient was a 43-year-old female and the second patient a 31-year-old female. Both patients had an history of splenectomy and had received occasionally blood transfusions. The right heart catheterization (RHC) revealed pre-capillary PH (mPAP: 31 mmHg, PAWP: 6 mmHg and PVR: 3,6 Wood Units for the first patient; mPAP: 46 mmHg, PAWP: 9 mmHg and PVR: 5,2 Wood Units for the second patient). The perfusion lung scan was without signs of Chronic Thromboembolic Pulmonary Hypertension. We treated the two patients with PAH-specific therapy (Bosentan) together with intensification of haemoglobinopathy-directed therapy (regular blood transfusions, chelation, hydroxyurea). Both patients presented significant clinical and haemodynamic improvement. For the first patient, we decided to stop Bosentan and continue only with the haemoglobinopathy-directed therapy. Two years later, she remains in NYHA-FC I and has normal haemodynamic parameters in the follow-up RHC. The second patient decided on her own to discontinue Bosentan and maintain the transfusion programme. One year later, her clinical status was severely deteriorated as well as the haemodynamics derived from the new RHC. Thereafter, double-specific PAH therapy including Bosentan and Sildenafil added to her treatment, followed by clinical and haemodynamic improvement. In conclusion, reporting these two cases with the same initial approach and different outcome, we highlight the multifactorial mechanisms of PH development in patients with TI, the importance of optimal chelation transfusion therapy and the need of PAH-specific therapy in some patients.

Gender-dependent effects of integrin-linked kinase inhibitor Cpd22 on severe experimental pulmonary hypertension

Dmitry A. Goncharov^*, Yuanjun Shen^*, Theodore Avolio, Arnab Ray, Evelyn Okorie, Ana L. Mora, Rebecca Vanderpool, Tatiana V. Kudryashova and Elena A. Goncharova

Pittsburgh Heart, Lung and Blood Vascular Medicine Institute, Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, Ageing Institute, Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA; Division of Translational and Regenerative Medicine, The University of Arizona College of Medicine, Tucson, AZ, USA

^*These authors contributed equally.

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by remodeling of small pulmonary arteries (PA), increased PA pressure (PAP), and death of heart failure. PAH affects predominantly females, but male patients develop more severe disease and respond poorer to current therapies. Integrin-linked kinase (ILK) supports PAH PA vascular smooth muscle cells (PAVSMC) proliferation and experimental PH in male mice, but effects of ILK inhibition on severe experimental PH in either males or females are unknown. We induced severe PH in male and female rats using single SU5416 injection (sq, 20 mg/kg) followed by three-weeks exposure to 10% normobaric hypoxia and two weeks of normoxia; vehicle or ILK inhibitor Cpd22 (20mg/kg, ip, 5 days/week) administrated at weeks 6–8. Results show that five weeks after PH induction, male and female rats developed similar levels of PH compared to controls. Eight weeks after PH induction, vehicle-treated male rats had more severe PH than females. Cpd22-treated male rats showed significantly reduced pulmonary vascular remodeling, RV hypertrophy and RV end-diastolic pressure, improved systolic right ventricular pressure (sRVP), PAP, and RV contractility index compared to vehicle-treated group. Cpd22-treated female rats showed no significant improvement. Analysis of proliferation (Ki67) of human male and female PAH PAVSMC treated with Cpd22 and 17β-estradiol separately or in combination revealed that 17β-estradiol, while having little effect as a single agent, significantly attenuated anti-proliferative effects of Cpd22 on the female, but not male PAH PAVSMC. Collectively, our data show that male rats develop more severe PH than females but respond better to Cpd22 treatment; observed resistance of females to Cpd22 may be explained, at least in part, by counter-effects of 17β-estradiol. Our findings highlight 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-remodeling effects of ILK inhibitors in females.

Involvement of secreted protein acidic and rich in cysteine in PASMC function in pulmonary hypertension

Christine Veith, Ipek Vartürk-Özcan, Simone Kraut, Jochen Wilhelm, Oleg Pak, Friederike C. Weisel, Karin Quanz, Monika Brosien, Andreas Günther, Werner Seeger, Ralph T. Schermuly and Norbert Weissmann

German Center for Lung Research, Excellence Cluster Cardio-Pulmonary Institute, Universities of Giessen and Marburg Lung Center, Giessen, Germany

Pulmonary hypertension (PH) is a severe disease, characterized by excessive pulmonary vascular remodeling, causing elevated pulmonary arterial pressure and right heart hypertrophy. Among other factors, PH can be triggered by chronic hypoxia, leading to pulmonary arterial smooth muscle cell (PASMC) hyper-proliferation. Upon re-exposure to normoxia, chronic hypoxia-induced PH in mice can be reversed. In the present study, we aim to identify novel candidate genes for pulmonary vascular remodeling and its reversal. C57BL/6J mice were exposed to normoxia (21% O₂), chronic hypoxia (10% O₂), or chronic hypoxia with subsequent re-exposure to normoxia for 1, 3, 7, and 14 days, followed by laser microdissection of pulmonary vessels. Microarray analysis identified secreted protein acidic and rich in cysteine (SPARC) as one gene consistently downregulated in all re-oxygenation time points investigated. SPARC was enhanced expressed in chronic hypoxic mice and in idiopathic pulmonary arterial hypertension patients. Immunohistochemical staining localized SPARC in the pulmonary vasculature, site of pulmonary vascular remodeling. Furthermore, transforming growth factor-β1 or hypoxia/hypoxia-inducible factor-2α signaling pathways induced SPARC expression in in vitro studies, in primary human PASMC. Moreover, SPARC silencing led to reduced PASMC proliferation and diminished phosphatidylinositol 3-kinase-Akt pro-proliferative signaling. In contrast, in hypoxic SPARC knockout PASMC, proliferation and Akt activation was enhanced. As expected, in in vivo experiments, SPARC knockout mice possessed a similar degree of hypoxia-induced PH than littermate controls. Our study indicates the involvement of SPARC in primary human PASMC proliferation and its association with human PH disease.

Senescent cells: role in countering pulmonary hypertension development

E. Born, M. Breau, S. Abid, A. Houssaini, D. Beaulieu, E. Marcos, R. Pierre, Cruzeiro M. Do, M. Delcroix, R. Quarck, L. Lipskaia and S. Adnot

INSERM U955 and Département de Physiologie, Hôpital Henri Mondor, Créteil, France; Université Paris-Est Créteil, Créteil, France; Respiratory Division, Department of Clinical and Experimental Medicine, University Hospitals of Leuven, University of Leuven, Leuven, Belgium

The role of senescent cells (SCs) in the pathogenesis of pulmonary hypertension (PH) remains undefined. Based on our finding in patients with pulmonary arterial hypertension (PAH) of abundant senescent p16- and p21-positive cells within remodeled pulmonary vessels and of high lung levels of p16, p21, and γH2AX proteins, we investigated SCs in experimental PH. We used p16^LUC/+ knock-in (p16^LUC/LUC) mice expressing luciferase in SCs to monitor luminescence imaging in vivo and assess p16 inactivation; p16^{GFP-INK-ATTAC} mice, carrying a p16 promoter-driven killer gene construct that can be pharmacologically activated in vivo to eliminate SCs; and wild-type mice treated with the senolytic drug ABT263. The mice were exposed to normoxia (N), chronic hypoxia (H), or H plus Sugen (H + S). ABT263 was also studied in monocrotaline (MCT)-treated rats. Results show that exposure to H was associated with a time-dependent increase in lung SCs as assessed by thoracic bioluminescence in p16^LUC/LUC, lung p16 and p21 protein levels, lung levels of senescence-associated secretory phenotype (SASP) components, genomic and telomeric DNA damage, telomere shortening, and oxidative stress parameters. Elimination of p16-positive SCs in p16^{GFP-INK-ATTAC} mice increased both right ventricular systolic pressure (RVSP) and vessel remodeling in young and old mice during N, H, and H + S. ABT263 treatment (50 mg/kg/day) increased RVSP during N and aggravated PH induced by H or H + S in wild-type mice. SC elimination was associated with SASP downregulation after 24 h and 21 days (RNA-Seq analysis) of H exposure. In rats, ABT263 increased pulmonary artery pressure and vessel remodeling and worsened MCT-induced PH. The p16^LUC/LUC mice with genetic p16 inactivation responded to N or H by increased RVSP and pulmonary vessel remodeling at 12–18 months but not at 4 months. In conclusion, counteracting cell senescence alters pulmonary hemodynamics in healthy animals and aggravates experimental PH.

Benefit of combination therapy over monotherapy for the treatment of pulmonary arterial hypertension according to risk profile: limitations of risk stratification in clinical practice

P.A. Aurtenetxe, O.C. Pérez, J.A. Barberá, M. López-Meseguer, R. López-Reyes, J.A. Domingo-Morera, E. Garrido-Lestache, Peñate G.M. Pérez, Soriano J. Rueda and P. Escribano-Subías

Pulmonary Hypertension Unit, Cardiology Department, Hospital Universitario 12 de Octubre, Madrid, Spain; Department of Pulmonary Medicine, Hospital Clínic, University of Barcelona, Barcelona, Spain; Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain; Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), Madrid, Spain; Pneumology Department, Hospital Universitari Vall d’Hebron, Barcelona, Spain; Pneumology Department, Hospital Universitario La Fe, Valencia, Spain; Pneumology Department, Hospital Universitario Miguel Servet, Zaragoza, Spain; Pediatric Cardiology Department, Hospital Universitario Ramón y Cajal, Madrid, Spain; Pulmonary Circulation Unit, Pneumology Department, Hospital Universitario de Gran Canaria Dr. Negrín, Las Palmas, Spain; Cardiology Department, Hospital Universitario La Fe, Valencia, Spain; Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBERCV), Madrid, Spain; Instituto de Salud Carlos III, Madrid, Spain; REHAP (Registro Español de Hipertensión Arterial Pulmonar) Investigators (Spanish Registry of Pulmonary Arterial Hypertension)

The assessment of prognosis of patients with pulmonary arterial hypertension (PAH) is considered an important part of care and the current treatment strategy is based on systematic assessment of clinical response. The multiparametric risk stratification approach is utilised for defining a low, intermediate or high-risk status. The aim of this study was to determine the impact of drugs approved for PAH in modifying the risk score of treatment-naïve PAH patients. This is a retrospective study of 371 newly diagnosed and treatment-naïve PAH type 1 patient (incident). Patients were enrolled in the REHAP registry between 1 January 2010 and 31 December 2017. Demographic, haemodynamic, clinical variables (WHO, heart failure symptoms), exercise (6MWT/CPET) and right ventricular function echo parameters were collected at the beginning of the treatment with specific treatment and at 3–12 months follow-up. Patients with suspicion or high probability of pulmonary veno-occlusive disease or uncorrected congenital heart disease were excluded. Results show that 65% were female, mean age 54 years. The most frequent aetiology was idiopathic and hereditary PAH (38%) followed by scleroderma (30%). At baseline, most of the patients were in intermediate risk (62%) and at follow-up 40% remains the same. At baseline in low and intermediate risk, the more frequent treatment is monotherapy and in the high-risk, the double combination. Survival rates were significantly higher in patients with lower risk, both at baseline and follow-up, and for those maintaining or achieving a low-risk profile at follow-up. Triple therapy was associated with poorer survival in patients who achieved/maintained a low-risk profile at follow-up. In conclusion, although the stratification strategy used found differences in survival rates according to risk of patients and demonstrated the benefit of achieving/maintaining a low-risk profile, we were unable to demonstrate the benefit of combination over monotherapy. Inability to properly discriminate the risk in populations with mixed aetiologies may account for this failure.

Treatment of severe precapillary pulmonary hypertension with intravenous prostanoids in patients with hereditary hemorrhagic telangiectasia

Stephanie Hon, Eric Abston and Harrison W. Farber

The Pulmonary Center, Boston University School of Medicine, Boston, MA, USA; Pulmonary, Critical Care & Sleep Division, Tufts Medical Center, Boston, MA, USA

Hereditary hemorrhagic telangiectasias (HHT) is characterized by arteriovenous malformations (AVM), an autosomal dominant inheritance pattern, and age-related penetrance. It is diagnosed clinically with three or more Curacao criteria: telangiectasias, epistaxis, family history, visceral lesions. Pulmonary hypertension (PH) occurs in 15% of patients with HHT, most frequently due to a high output state associated with hepatic AVM. In addition, a small, but significant, portion of patients with HHT develop precapillary PH, which portends poorer outcomes than for patients with HHT alone. To date, only small case series of treatment of severe precapillary PH in patients with HHT exist. Among these series, there has been no experience with prostanoid therapy as a treatment for HHT-associated PH, possibly because of the theoretical increased risk of bleeding in these patients due to the anti-platelet effect of these drugs. The purpose of this report is to emphasize the potential role and safety of prostanoid therapy as treatment for severe precapillary PH in patients with HHT. We retrospectively evaluated four patients with HHT and severe precapillary PH, treated with prostanoid therapy. Diagnostic hemodynamics were compared pre- and post-treatment using a paired t-test. Mean follow-up was 95 months. All subjects demonstrated clinically significant improvement following treatment with prostanoid therapy (epoprostenol). Cardiac index improved from 2.2 ± 0.8 L/min/m² at baseline to 3.6 ± 0.5 L/min/m² during therapy. Pulmonary vascular resistance decreased from 16 ± 6 WU to 7 ± 1 WU. Mean pulmonary arterial pressure decreased from 68 ± 6 mmHg to 53 ± 9 mmHg. Moreover, treatment with epoprostenol was safe; there were no clinically important bleeding episodes associated with therapy in these patients. This is the first series to examine prostanoid therapy as treatment of severe HHT-associated precapillary PH. All patients treated with this regimen demonstrated hemodynamic improvement and there were no unexpected safety issues. Further studies are warranted to evaluate this treatment regimen for HHT-associated PH.

Molecular characterization of pulmonary vasculature development

Stephanie Hon, Anna Engler, Carlos V. Martin, Hailey Heston and Jason Rock

Center of Regenerative Medicine (CReM), Boston University, Boston, MA, USA

The temporospatial development of the pulmonary vasculature is poorly understood. A better understanding of the cellular interactions that specify normal development of the pulmonary vasculature could lead to new therapies for conditions such as pulmonary hypertension. We aim to characterize vascular cells in murine embryonic lungs as they progress through developmental stages. Cdh5-CreER ROSA-TdTm mice were injected with tamoxifen at E8.5, to mark the progeny of early vascular progenitor cells by genetic lineage tracing. Lungs were harvested at E14.5 and analyzed by immunofluorescence staining, FACS analysis, and single-cell RNA sequencing. We detected TdTm+ cells with morphologies and distributions consistent with venous, arterial, and capillary endothelial cells. This suggests that there is a multipotent vascular precursor population as early as E8.5. Tm4sf1 was identified as a potential marker of multipotent vascular precursors in single-cell transcriptomic data. Our objective is to further characterize the expression of Tm4sf1 in these progenitor cells and its role in the development of mature pulmonary vasculature.

Pulmonary arterial hypertension in pregnancy: a systematic review of adverse outcomes in the modern era

Ting T. Low, Nita Guron, Robin Ducas, Pradeepkumar Charla, Kenichiro Yamamura, Rohan D’ Souza, John Granton and Candice K. Silversides

National University Heart Centre, Singapore; Toronto General Hospital, Toronto, Canada; Mt Sinai Hospital, Toronto, Canada

Pulmonary arterial hypertension (PAH) in pregnancy is a perilous clinical situation, known historically to result in high maternal mortality. With advances in PAH therapy and specialised care, there may be more favourable pregnancy outcomes in the modern era. We sought to systematically evaluate the adverse outcomes of PAH in pregnancies in the last decade. We searched Medline, Embase and Cochrane databases for articles describing outcomes in pregnancy cohorts published between 2008 and 2018. We excluded studies with less than five cases and those with insufficient data on follow-up or PAH therapy. Two reviewers independently screened 3658 titles and included 13 studies for analysis. Pooled incidences and percentages of maternal and perinatal outcomes were calculated. Results showed that there were 272 pregnancies reported in 258 women, 217 pregnancies advanced beyond 20 weeks gestation. The mean maternal age was 28 ± 2 years, mean pulmonary artery systolic pressure on echocardiogram was 76 ± 19 mmHg, 74% was in good functional class I and II. The aetiologies include idiopathic PAH 21% (n = 46), congenital heart disease 63% (n = 136) and other aetiologies 15% (n = 37). Only 48% of patients received targeted PAH therapy in pregnancy, of whom 17% had combination therapy. Premature births occur in 58% of pregnancies, with a mean gestation age of 34 ± 1 weeks at delivery. Perinatal mortality was 4% (n = 8); 3% stillbirths and 1% neonatal deaths. Maternal mortality rate for advanced pregnancies was 12% overall (n = 26); by aetiology breakdown, it was 20% for idiopathic PAH (9/46), 11% for congenital heart disease (15/136) and 6% for other aetiology (2/32). The reported causes of death included right heart failure, cardiac arrest, PAH crises, pre-eclampsia and sepsis. The immediate post-partum period was most precarious, 61% of maternal deaths occur at 0–4 days post-partum. PAH in pregnancy continues to be hazardous with high maternal mortality rate. Continued prospective studies are needed to improve outcomes.

Endothelial C-terminal Src kinase is associated with arteriolar fibrosis and impaired right ventricle-pulmonary artery coupling in early-stage pulmonary arterial hypertension

Bradley M. Wertheim, Rui-sheng Wang, Ying-Yi Zhang, Andriy O. Samokhin, George A. Alba, Elena Arons, Paul B. Yu and Bradley A. Maron

Division of Cardiovascular Medicine, Pulmonary and Critical Care Medicine, Department of Medicine, Channing Division of Network Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA; Division of Pulmonary and Critical Care Medicine, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA

Pulmonary arterial hypertension (PAH) is characterized by vascular fibrosis and right ventricular (RV) failure. The role of oxidant stress in advanced PAH is well-established; however, the pathogenetic implications of pro-fibrotic redox pathways in early-stage PAH are unknown. We hypothesized that unique redox-sensitive molecular targets in pulmonary artery endothelial cells (PAECs) distinguish early- from advanced-stage PAH. Sprague-Dawley rats were administered monocrotaline (MCT) (60 mg/kg) (day 0) and invasive hemodynamics, histopathology, and PAEC transcriptomics were analyzed on days 15 and 23 for early-stage and advanced PAH, respectively. Results showed that compared to untreated control, early-stage PAH demonstrated impaired RV–PA coupling (1.13 ± 0.05 vs. 0.90 ± 0.06 Ees/Ea, P = 0.02), increased indexed pulmonary vascular resistance (50 ± 8 vs. 213 ± 29 mmHg*min*g⁻¹*mL⁻¹, P<0.001), and a 2.7-fold increase in arteriolar collagen by picrosirius stain (P < 0.0001) without substantial pulmonary hypertension (25 ± 1.2 vs. 33.2 ± 2.7 mmHg PA systolic pressure, P = 0.02). The PAEC transcriptome in early-stage MCT-PAH included N = 1058 unique, differentially expressed genes (FDR < 0.05), which was enriched for fibrosis genes (P = 0.019). These data were used to develop a novel protein–protein interaction (PPI) network for predicting mediators of endothelial fibrosis in early-stage PAH. Dynamic network biomarker and gene ontology analyses suggested PPIs involving c-terminal src kinase (Csk) are important in early-stage PAH fibrosis in silico. Compared to control, Csk mRNA (log fold-change = +0.72; FDR < 0.0001) and protein expression (123 ± 12 vs. 260 ± 25, a.u., P = 0.01) were increased in PAECs in early-stage PAH by RNAseq ex vivo and anti-Csk immunofluorescence in situ, respectively. Compared to control, hydrogen peroxide (250 µM) treatment increased PAEC Csk by 2.1-fold (P = 0.02). Furthermore, Csk correlated positively with arteriolar collagen quantity in MCT-PAH (r = +0.87, P = 0.006). Collectively, early-stage PAH is defined by a unique endothelial pathobiology relative to advanced PAH. Redox-regulation of endothelial fibrosis by Csk may have important therapeutic implications for early-stage PAH.

Long noncoding RNA TYKRIL plays a role in pulmonary hypertension via the p53-mediated regulation of PDGFRβ

Christoph M. Zehendner^*, Chanil Valasarajan^*, Astrid Werner, Jes-Niels Boeckel, Florian C. Bischoff, David John, Tyler Weirick, Simone F. Glaser, Oliver Rossbach, Nicolas Jaé, Shemsi Demolli, Ke Yuan, Jesus Perez Vinicio A. de, Katharina M. Michalik, Wei Chen, Werner Seeger, Andreas Guenther, Roxana M. Wasnick, Shizuka Uchida, Andreas M. Zeiher, Stefanie Dimmeler^# and Soni S. Pullamsetti^#

Department of Cardiology, Goethe University, Frankfurt am Main; German Center for Cardiovascular Research, DZHK, Berlin; Department of Lung Development and Remodeling, Max Planck Institute for Heart and Lung Research, member of the German Center for Lung Research (DZL), Bad Nauheim; Department of Biology and Chemistry, University of Giessen, Giessen; Laboratory for Novel Sequencing Technology, Max-Delbruck-Centre for Molecular Medicine, Berlin; Department of Internal Medicine, Universities of Giessen and Marburg Lung Center (UGMLC), member of the DZL, Justus Liebig University, Giessen, Germany; Cardiovascular Innovation Institute, University of Louisville, Louisville, KY; Division of Pulmonary and Critical Care Medicine, Stanford University, Stanford, CA, USA; and Department of Biology, Southern University of Science and Technology, Shenzhen, Guangdong, China

^*These first authors contributed equally.

^#These senior authors contributed equally.

Pulmonary hypertension (PH) is characterized by pathological remodeling of pulmonary vessels and elevated pulmonary pressures. The pro-proliferative and anti-apoptotic phenotype of various resident and non-resident cells contribute to the vessel remodeling and molecular mechanism driving this phenotype is not well understood. Long noncoding RNAs (lncRNAs) are emerging as important regulators of diverse biological functions. Their role in pulmonary arterial hypertension (PAH) remains to be explored. From the RNAseq data of pericytes and pulmonary arterial smooth muscles cells (PASMCs) exposed to hypoxia and derived from idiopathic PAH (IPAH) patients, a novel lncRNA TYKRIL was identified to be consistently upregulated. TYKRIL knockdown in pericytes and PASMCs reversed the pro-proliferative and anti-apoptotic phenotype induced under hypoxic and IPAH conditions. Overexpression of TYKRIL using dCAS-VP64 system in control pericytes induced pro-proliferative and anti-apoptotic phenotype. Due to the poor species conservation, ex-vivo studies were carried out in precision cut lung slices (PCLS) from PH patients. Knockdown of TYKRIL in PCLS significantly reduced vascular remodeling and decreased the proliferating cell nuclear antigen (PCNA)-positive cells in the vessels. From the RNAseq analysis, it was observed that TYKRIL knockdown downregulated expression of various receptor tyrosine kinases. Expression of PDGFRβ, a key player in PH, was found to be strongly correlated to TYKRIL expression in the patient samples. From the transcription factor-screening array, it was observed that TYKRIL knockdown increased the p53 activity, a known repressor of PDGFRβ. Using RNA immunoprecipitation (RIP), it was found that TYKRIL interacts with p53. iCLIP Individual-nucleotide resolution UV crosslinking and immunoprecipitation and RIP using various p53 mutants demonstrated that the first exon of TYKRIL binds to the N-terminal of p53 (an important region for p300 interaction with p53). The proximity ligation assay reviled that TYKRIL interferes with the p53-p300 interaction and regulates p53 nuclear translocation. In conclusion, TYKRIL plays an important role in PH by regulating the p53/PDGFRβ axis.

PET imaging reveals early pulmonary perfusion abnormalities in HIV infection similar to smoking

Puja Kohli, Vanessa J. Kelly, Kathryn A. Hibbert, BjÖrn Corleis, Mamary Kone, Josalyn Cho, Doreen DeFaria-Yeh, Douglas S. Kwon, Benjamin Medoff, R.S. Harris and Tilo Winkler

Division of Pulmonary and Critical Care, Massachusetts General Hospital, Boston, MA, USA; Department of Medicine, Harvard Medical School, Boston, MA, USA; The Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA; Department of Internal Medicine and Carver College of Medicine, The University of Iowa, Iowa City, IA, USA; Division of Cardiology, Division of Infectious Disease, Department of Anesthesia, Massachusetts General Hospital, Boston, MA, USA

COPD is the most common non-infectious pulmonary disease among people living with HIV, independent of smoking. However, the cause for this enhanced susceptibility remains unclear, and the effects of HIV on pulmonary perfusion are unknown. We used PET-CT in 46 smokers and non-smokers, 23 of whom had with documented HIV infection. Emphysema was assessed by CT and perfusion by nitrogen-13 PET scans. After removal of image noise, vertical and axial gradients in perfusion were calculated. We tested for differences in the total spatial heterogeneity of perfusion (CV²Qtotal) and its components (CV²Qtotal = CV²Qvgrad (vertical gradient) + CV²Qzgrad (axial gradient) + CV²Qr (residual heterogeneity)) among groups. Results showed that there were no significant differences in demographic parameters among groups, and all subjects had minimal radiographic evidence of emphysema. Compared to controls, non-smokers living with HIV had a significantly greater CV² Qr/CV²Qtotal (0.48 vs. 0.36, p = 0.05) and reduced CV²Qvgrad/CV²Qtotal (0.46 vs. 0.65, p = 0.038). Smokers also had a reduced CV²Qvgrad/CV²Qtotal, however, there was no significant difference in CV²Qvgrad/CV²Qtotal between smokers living with and without HIV (0.39 vs. 0.34, p = 0.58), despite a decreased vertical perfusion gradient (Qvgrad) in smokers living with HIV. In conclusion, in non-smokers living with well-controlled HIV and minimal radiographic emphysema, HIV infection contributes to pulmonary perfusion abnormalities similar to smokers. These data indicate the onset of subclinical pulmonary perfusion abnormalities that could herald the development of significant lung disease in these susceptible individuals.

Perfusion imaging distinguishes exercise pulmonary arterial hypertension at rest

Puja Kohli, Vanessa J. Kelly, Ekaterina G. Kehl, Josanna Rodriguez-Lopez, Kathryn A. Hibbert, Mamary Kone, David M. Systrom, Aaron B. Waxman, Jose G. Venegas, Richard Channick, Tilo Winkler^* and R.S. Harris^*

Division of Pulmonary and Critical Care Medicine, Division of General Internal Medicine, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA; Division of Pulmonary and Critical Care Medicine, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA; Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA

^*These senior authors contributed equally.

Whether exercise pulmonary arterial hypertension (ePAH) is a distinct disease, or a mild form of pulmonary arterial hypertension (PAH), remains uncertain. We aimed to test the hypothesis that pulmonary perfusion imaging would demonstrate resting pulmonary perfusion abnormalities in ePAH subjects on a spectrum between healthy controls and PAH subjects. Subjects underwent high-resolution CT followed by ¹³NN-saline functional PET imaging of pulmonary perfusion at baseline and while breathing inhaled nitric oxide and oxygen (O₂ + iNO). The total spatial heterogeneity of perfusion (CV²Qtotal) and the heterogeneity caused by its components (CV²Qvgrad, vertical gradient; CV²Qzgrad, axial gradient; CV²Qr, residual heterogeneity) were assessed at baseline and while breathing O₂ + iNO. We studied five healthy controls (HC), four ePAH, and four PAH subjects. Results showed that ePAH subjects had greater CV²Qtotal than both PAH subjects and HC at baseline, with further discrimination while breathing O₂ + iNO (3.4-fold, p = 0.0009 and 1.71-fold, p = 0.009, respectively). ePAH subjects also had greater CV²Qr and perfusion heterogeneity across different image resolutions (10–110 mm) (p < 0.01 by ANOVA for all filter sizes). ePAH subjects and HC had similar vertical gradients in perfusion (Qvgrad) at baseline with similar gradient amplifications while breathing O₂ + iNO. PAH subjects, on the other hand, had minimal Qvgrad at baseline and no substantial change while breathing O₂ + iNO. In conclusion, ePAH subjects have an abnormal regional perfusion pattern at rest that is distinct from both PAH subjects and healthy controls, suggesting that ePAH may represent a distinct clinical entity from PAH although longitudinal studies are required.

The combination of metabolomic and miRNA diagnostic scores to identify pulmonary hypertension and sub-classify pulmonary arterial hypertension

Christopher J. Rhodes, Timothy Jatkoe, John Wharton, Dennis Wang, Niamh Errington, Charles Bridges, Yiu-Lian Fong, Mark Toshner, Luke S. Howard, Allan Lawrie and Martin R. Wilkins

Imperial College, London, UK; Jannsen, Johnson & Johnson Innovations, New Brunswick, NJ, USA; University of Sheffield, Sheffield, UK; University of Cambridge, Cambridge, UK

The patient journey from first symptoms to a diagnosis of pulmonary hypertension (PH) typically takes 1–3 years, and despite increased awareness, therapeutic advances and improved outcomes, this has remained unchanged over the last 20 years. PH is characterised by disturbed metabolic function and altered levels of circulating miRNA such as miR-150 have been associated with pulmonary arterial hypertension (PAH, group 1 PH) and survival. In systemic sclerosis, screening algorithms to identify patients earlier can improve outcome but remain resource intensive. Early diagnosis and classification of PH and PAH could be assisted by simple non-invasive testing of circulating metabolites and miRNA. Serum from 1521 patients with PH and disease comparators (chronic thromboembolic disease and patients referred to PH clinics who had PH ruled out) and healthy controls were collected from three UK expert centres (Imperial, Papworth and Sheffield) and analysed for 1523 metabolites (Metabolon) and 554 miRNAs (MiRXES). In a discovery–validation design, molecules with classifying profiles in PH and PAH patients were identified, and diagnostic models constructed. Prognosis was also assessed in PAH patients divided by miR-150 levels. Results showed that a LASSO model of five miRNA with age, sex, BMI and eGFR distinguished PAH from other PH groups and disease comparators. Metabolomic profiles were able to differentiate all PH groups from disease comparators and healthy controls in univariate analyses and LASSO modelling achieved an AUC of 0.84. The combination of these models was able to distinguish both PH from controls and PAH from other PH groups. Low miR-150 levels identified PAH patients with poor survival outcomes. In conclusion, high-throughput profiling of circulating metabolites and miRNA enabled construction of models capable of distinguishing PH patients from relevant comparator groups and differentiate subgroups of PH. Prospective studies validating the clinical utility of these models in clinical settings early in the diagnostic pathway are required.

Assessment of the REPLACE study composite endpoint in the PATENT study and association with long-term outcomes in riociguat-treated patients

G. Simonneau, M.M. Hoeper, P.A. Corris, S. Rosenkranz, E. Grünig, J. White, D. Langleben, C. Meier, D. Busse, F. Kleinjung and R.L. Benza

Service de Pneumologie, Assistance Publique–Hôpitaux de Paris, Hôpital Bicêtre, Université Paris-Sud, Laboratoire d’Excellence en Recherche sur le Médicament et Innovation Thérapeutique, and INSERM Unité 999, Le Kremlin-Bicêtre, France; Clinic for Respiratory Medicine, Hannover Medical School, Hannover, Germany, Member of the German Center for Lung Research; Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK; Department III of Internal Medicine, Cologne University Heart Center, Cologne, Germany; Centre for Pulmonary Hypertension at Thoraxklinik Heidelberg, Heidelberg, Germany; Division of Pulmonary and Critical Care Medicine and the Mary M. Parkes Center, University of Rochester Medical Center, Rochester, NY, USA; Center for Pulmonary Vascular Disease and Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, Canada; Bayer AG, Berlin, Germany; Chrestos Concept GmbH & Co. KG, Essen, Germany; Cardiovascular Institute, Allegheny General Hospital, Pittsburgh, PA, USA

REPLACE will evaluate switching to riociguat in patients with pulmonary arterial hypertension (PAH) and an insufficient response to phosphodiesterase-5 inhibitors. The primary outcome is a composite clinical improvement endpoint (no clinical worsening and two of: ≥10% or ≥30 m improvement in six-minute walking distance; World Health Organization functional class I/II; ≥30% decrease in N-terminal pro-brain natriuretic peptide). We assessed whether the endpoint could differentiate between the riociguat and placebo arms of the PATENT-1 study, and whether achieving the endpoint was associated with long-term outcomes. In PATENT-1, patients received placebo or riociguat up to 2.5 mg three times/day (tid). In PATENT-2, former placebo patients received riociguat up to 2.5 mg tid. This post-hoc analysis applied the REPLACE composite endpoint to the PATENT-1 database and evaluated the association between achieving the composite endpoint in PATENT-1 and survival and clinical worsening-free survival in PATENT-2. Results showed that the PATENT-1 analysis included 126 patients who received placebo and 254 who received riociguat up to 2.5 mg tid, with long-term outcomes assessed in 340 patients who entered PATENT-2. At Week 12, 50% of riociguat-treated and 25% of placebo-treated patients achieved the composite endpoint (odds ratio, 3.13; 95% confidence interval, 1.88–5.26; p < 0.001). Similar results were observed irrespective of background therapy, although more treatment-naïve patients achieved the endpoint than pretreated patients in the riociguat arm. Patients who achieved the endpoint in PATENT-1 had a 45% reduction in relative risk of death and a 19% reduction in relative risk of clinical worsening in PATENT-2 compared with those who did not. In conclusion, in this post-hoc analysis of PATENT-1 data, riociguat-treated patients were more likely than placebo-treated patients to achieve the REPLACE composite endpoint of improvement, which was associated with improved long-term outcomes. Use of this endpoint in patients with PAH is a viable assessment of treatment response.

Skeletal muscle LKB1/SIRT3-AMPK-GLUT4 activation by treprostinil and metformin normalizes hyperglycemia and improves cardiac function in pulmonary hypertension associated with heart failure with preserved ejection fraction (PH-HFpEF)

Longfei Wang, Gunner Halliday, Joshua R. Huot, Taijyu Satoh, Jeff J. Baust, Jian Hu, Amanda Fisher, Rebecca R. Vanderpool, Robert V. Considine, Andrea Bonetto, Jiangning Tan, Timothy N. Bachman, Andrea Sebastiani, Ana L. Mora, Mark T. Gladwin and Yen-Chun Lai

Pittsburgh Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA, USA; The Third Xiangya Hospital, Central South University; Changsha, China; Division of Pulmonary, Critical Care, Sleep and Occupational Medicine; Division of Endocrinology, Department of Surgery, Indiana University, Indianapolis, IN, USA; Department of Cardiovascular Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan; Division of Translational and Regenerative Medicine, University of Arizona, Tucson, AZ, USA

Pulmonary hypertension associated with heart failure with preserved ejection fraction (PH-HFpEF; Group 2) is the most common cause of PH worldwide but has no proven drug therapy at present. PH-HFpEF is associated with insulin resistance and features of metabolic syndrome. The stable and long-lasting prostacyclin analog, treprostinil, is an effective and widely used FDA-approved drug for the treatment of pulmonary arterial hypertension. While the effect of treprostinil on metabolic syndrome is unknown, a recent study suggests that prostacyclin analog beraprost can improve glucose intolerance and insulin sensitivity. We sought to evaluate the effectiveness of treprostinil in the treatment of metabolic syndrome-associated PH-HFpEF. Treprostinil treatment was given to mice with mild metabolic syndrome-associated PH-HFpEF induced by high-fat diet (HFD) and to SU5416/Obese ZSF1 rats, a model which is created by the treatment of rats with a more profound metabolic syndrome due to double leptin receptor defect (obese ZSF1) with a vascular endothelial growth factor receptor blocker SU5416. Results showed that in HFD-exposed mice, chronic treatment with treprostinil prevented hyperglycemia and pulmonary hypertension. In SU5416/Obese ZSF1 rats, treprostinil improved hyperglycemia with similar efficacy to that of metformin—the first-line drug for type 2 diabetes; the glucose-lowering effect of treprostinil was further potentiated by the combined treatment with metformin. Early treatment with treprostinil in SU5416/Obese ZSF1 rats lowered pulmonary pressures and a late treatment with treprostinil together with metformin improved pulmonary artery acceleration time to ejection time ratio and tricuspid annular plane systolic excursion with robust activation of skeletal muscle LKB1/SIRT3-AMPK-GLUT4 signaling. In conclusion, our data suggest a potential role of treprostinil as a preventative treatment for mild metabolic syndrome-associated PH-HFpEF. Our data also suggest that combined treatment with treprostinil and metformin may be a potential effective therapy for a more severe disease.

Circulating long non-coding RNA H19 as a novel biomarker for right ventricular failure associated with pulmonary arterial hypertension

Junichi Omura, Karima Habbout, Tsukasa Shimauchi, Sandra Breuils-Bonnet, Eve Tremblay, Sandra Martineau, Valérie Nadeau, François Potus, Stephen L. Archer, Allan Lawrie, Roxane Paulin, Steeve Provencher, Olivier Boucherat and Sébastien Bonnet

Pulmonary Hypertension and Vascular Biology Research Group of Quebec Heart and Lung Institute, Laval University, Quebec, Canada; Department of Medicine, Queens University, Kingston, Canada; Department of Infection, Immunity and Cardiovascular Disease, University of Sheffield, Sheffield, UK

Right ventricular failure (RVF) is a major adverse prognostic factor in pulmonary arterial hypertension (PAH). Recent omics analyses demonstrated the deregulation of long non-coding RNAs (LncRNAs) in cardiovascular disease. Moreover, circulating LncRNAs have been considered as novel biomarkers for diagnosis and prognosis of cardiovascular diseases. The LncRNA H19 has been implicated in cardiac hypertrophy and fibrosis but its role in RVF remains unknown. In human RV from control donors, patients with compensated RV hypertrophy (cardiac index (CI) >2.2) and decompensated RV (dRV, PAH patients that died from RVF), H19 was specifically up-regulated in dRV. Similar findings were noted in several RVF rodent models. We exploited an existing cohort from Canada composed of patients with idiopathic PAH (IPAH, n = 52) and controls (n = 57) and measured H19 expression in plasma. We sought replication in an independent validation cohort of subjects with IPAH (n = 75) and controls (n = 54) from UK. H19 plasma levels measured by qRT-PCR were significantly elevated and correlated with RVF severity in IPAH from each cohort respectively or combined cohorts. Moreover, H19 levels in plasma were predominantly increased in IPAH patients with dRV (CI < 2.2) in each cohort respectively or combined cohorts. Interestingly, H19 was higher and distinguished IPAH with the future occurrence of adverse events (death or lung transplant) in each cohort respectively or combined cohorts. Event-free survival estimates at one, three and five years were 98%, 92% and 84% for patients with H19 levels below 37.2 units versus 85%, 54% and 46% for those with H19 levels above 37.2 units (P < 0.001) when both cohorts were combined. Furthermore, the combination of plasma H19 and NT-proBNP levels or ERS/ESC risk score demonstrated better risk stratification of IPAH patients than either value alone. In conclusion, H19 circulating levels are a biomarker of RV function and prognosis in IPAH.

Long non-coding RNA H19 exacerbates right ventricular failure in PAH

Junichi Omura, Karima Habbout, Tsukasa Shimauchi, Sandra Breuils-Bonnet, Eve Tremblay, Sandra Martineau, Valérie Nadeau, François Potus, Stephen L. Archer, Jianhui Lin, Hamza Zafar, David G. Kiely, Allan Lawrie, Roxane Paulin, Steeve Provencher, Olivier Boucherat and Sébastien Bonnet

Pulmonary Hypertension and Vascular Biology Research Group of Quebec Heart and Lung Institute, Laval University, Quebec, Canada; Department of Medicine, Queen’s University, Kingston, Canada; Department of Infection, Immunity and Cardiovascular Disease, University of Sheffield, Sheffield, UK; Sheffield Pulmonary Vascular Disease Unit, Royal Hallamshire Hospital, Sheffield, UK

Right ventricular failure (RVF) is a major adverse prognostic factor in pulmonary arterial hypertension (PAH). Recent omics analyses have demonstrated the deregulation of several long non-coding RNAs (LncRNAs) in heart failure, but their role in RVF remains unknown. The LncRNA H19 and its encoded miR-675 have been implicated in both hypertrophic and fibrotic processes (two features of RVF) but never been studied in RVF. By qRT-PCR, we showed in human RV biopsies obtained from control donors, patients with compensated RV hypertrophy (cRV, cardiac index > 2.2) and decompensated RV (dRV, PAH patients died from RVF), that H19/miR-675 were specifically up-regulated in dRV (p < 0.001, n = 10–12/group) and correlated with cardiomyocyte hypertrophy and cardiac fibrosis. H19/miR-675 up-regulation was RV specific as no change was seen in either the LV or lung of PAH patients. Similar findings were noted in the monocrotaline (MCT) and the pulmonary artery banding (PAB) rat models of RVF, in which H19 expression inversely (p < 0.001) correlated with RV function, as evaluated by echocardiography and right heart catheterization; and positively (p < 0.001) with cardiomyocyte hypertrophy and cardiac fibrosis on histology. Knockdown of H19 using GapmeR improved RV function and reduced cardiomyocyte hypertrophy and cardiac fibrosis in MCT and PAB rats. Mechanistically, reduced expression of H19 was associated with upregulation of the epigenetic regulator EZH2, an established suppressor of cardiac hypertrophy and fibrosis. In vitro, inhibition of H19 suppressed phenylephrine (PE)-induced hypertrophy in both RV neonatal rat cardiomyocytes and H9C2 and upregulated EZH2. In contrast, forced expression of H19 using adenoviral vector induced cell hypertrophy. Consistent with these findings, EZH2 inhibition using siRNA induced cardiomyocyte hypertrophy under basal conditions, whereas its overexpression prevented PE-induced hypertrophy. In conclusion, our findings identify H19 as a new therapeutic target for the prevention of the transition from cRV to dRV in PAH.

Do plasma metabolomic profiles differentiate responders from nonresponders to oral treprostinil? Results from the FREEDOM phase 3 clinical development program

Christopher Rhodes, Derek Solum, Andrew Nelsen and Sudarshan Rajagopal

Imperial College London, London, UK; United Therapeutics Corporation, RTP, NC, USA; Duke University Hospital, Durham, NC, USA

Oral treprostinil (TRE) improves exercise capacity and delays disease progression in patients with pulmonary arterial hypertension (PAH). However, the tolerance and response to TRE varies between subjects. The objective of this study was to profile plasma of subjects with PAH who were treated with TRE or placebo to identify a metabolic signature that could predict TRE responders and provide insight into the metabolic changes induced by treatment. FREEDOM-C2 (TDE-PH-308/NCT00887978) was a 16-week, placebo-controlled, efficacy and safety study of oral treprostinil in PAH that measured change in median six-minute walk distance (6MWD). FREEDOM-EV (TDE-PH-310/NCT01560624) was an event-driven, placebo-controlled study of oral treprostinil in PAH that assessed time to clinical worsening. Subjects consented to participate in an optional biomarker substudy. Plasma was collected at Baseline and Week 16 for TDE-PH-308 (N = 187) and at Last Visit for TDE-PH-310 (N = 74). Based on improvement in 6MWD, subjects were classified as either responders or nonresponders. Samples were evaluated using a comprehensive metabolomic platform (Metabolon) with the potential to identify up to 4000 chemically and structurally diverse metabolites across a variety of classes. Results showed that metabolomic profiling detected 1346 distinct biochemicals. Responders from FREEDOM-C2 had altered metabolites associated with hypoxia, glycemic health, microbiome, and redox status; changes in the microbiome and redox status occurred post-treatment. For FREEDOM-EV, metabolites classes included clotting factors, xanthine metabolites, microbial products, and homoarginine decreased in responders versus nonresponders while metabolite classes that increased in responders included acetoacetate, fatty acids, and homocitrulline. In conclusion, these results revealed metabolites that distinguished between TRE responders and nonresponders. Metabolites involved in hypoxia, glycemic control, lipid metabolism, and microbial activity were generally correlated with improved clinical response. Subjects treated with TRE had distinct metabolic signatures associated with microbial architecture, activity, and host redox status.

Twist1 in hypoxia-induced vascular remodeling

Akiko Mammoto and Tadanori Mammoto

Departments of Pediatrics, Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, WI, USA

Remodeling of distal pulmonary arterioles (PAs) associated with accumulation of pulmonary artery smooth muscle cells (PASMCs) represents one of the major pathologic features of pulmonary hypertension (PH). We have reported that the transcription factor, Twist1 in ECs contributes to hypoxia-induced PH. Here we found that Twist1 overexpression increases the expression of platelet-derived growth factor (PDGFB) in cultured human pulmonary arterial endothelial (HPAE) cells. Conditioned media collected from Twist1 overexpressing HPAE cells induce PASMC migration and proliferation. Hypoxia upregulates the levels of Twist1 and PDGFB in HPAE cells. When we implant fibrin gel supplemented with endothelial cells (ECs) on the mouse lung, these ECs form vascular lumen structures and hypoxia stimulates accumulation of αSMA-positive cells to blood vessels and upregulates PDGFB expression in the gel, while Twist1 knockdown in ECs suppresses the effects. The levels of Twist1 and PDGFB are higher in PAE cells isolated from idiopathic pulmonary arterial hypertension (IPAH) patients compared to those from healthy controls. IPAH patient-derived PAE cells stimulate accumulation of αSMA-positive cells to blood vessels in the implanted gel, while knockdown of Twist1 inhibits the effects. In conclusion, these findings suggest that endothelial Twist1-PDGFB signaling plays a key role in SMC accumulation to PAs. Modulation of Twist1-PDGFB signaling could be an effective strategy for PH.

Novel BMPR2 mutation in hereditary pulmonary arterial hypertension

Sarah Chalmers, Stephen J. Murphy, James B. Smadbeck, George Vasmatzis and Mark E. Wylam

Division of Pulmonary and Critical Care Medicine, Mayo Clinic, Rochester, MN, USA; Molecular Medicine and Biomarker Discovery Program, Center for Individualized Medicine, Mayo Clinic, Rochester, MN, USA

Mutations in the bone morphogenic protein receptor two (BMPR2) gene occur in approximately 80% of cases of hereditary pulmonary arterial hypertension. Herein, we present a novel structural change in the BMPR2 gene identified by mate-pair sequencing (MPseq). Thirty-six-year-old female with HPAH presented for evaluation. Family history was pertinent for a sister, mother, cousin first removed, and second cousin with HPAH. Targeted sequencing and duplicate/deletion studies, including BMPR2, did not identify a causative mutation. She underwent heart and lung transplant and a novel donor vs. recipient cell-free DNA quantitative monitoring of junction sequences was used to interrogate graft health. Recipient pretransplant MPseq revealed a balanced inversion of a 5.7 Mb segment of chromosome 2 with break points within intron 10 of BMPR2 and intron 6 of PIKFYVE. Additionally, MPseq was performed on her sister and maternal cousin first removed and demonstrated a synonymous mutation. Gene mutation identification allows for informed genetic counseling regarding carrier testing prior to pregnancy, early identification of at-risk family members, and in the future, may allow for gene-targeted therapy. Heterozygous mutation in the BMPR2 gene is the most common mutation associated with HPAH. Current screening was blind to this BMPR2 genetic variant due to the copy neutral structure of the inversion. Mate-pair sequencing is a technique that utilizes long-insert paired-end DNA libraries followed by whole genome, paired-end next-generation sequencing and combines genome wide copy number analysis with nucleotide level resolution of breakpoint junctions to provide complete structural variant analysis and detection of the wide spectrum of genomic rearrangements. Thus, it allows for detection of small deletions, gains, and transposons which are not detected by standard mutational analysis. In our patient, identification of discordant fragments indicated an inversion. In conclusion, we suggest that MPseq be considered in all patients with HPAH without identifiable mutation.

The role of mitochondrial p66shc in biological aging of the lung

Claudia G. Castro, Natascha Sommer, Mariola Bednorz, Bakyt Kojonazarov, Simone Kraut, Stefan Hadzic, Nasim Alebrahimdehkordi, Stefan Arnhold, Rainer Schulz, Norbert Weissmann and Oleg Pak

Excellence Cluster Cardio-Pulmonary System (ECCPS), University of Giessen and Marburg Lung Center, Member of the German Center for Lung Research (DZL), Giessen, Germany; Veterinary Institute of Anatomy-Histology and Embryology, Giessen, Germany; Physiology Institute, Justus-Liebig-University Giessen, Giessen, Germany

During aging, the lung undergoes structural and functional changes associated with a decrease in lung function. It has been proposed that mitochondrial dysfunction and the accumulation of reactive oxygen species plays an important role during the aging process. The mitochondrial adaptor protein p66shc is a key regulator of mitochondrial reactive oxygen species, apoptosis, and aging. Thus, we investigated the role of p66shc in the aging process of the lung. At different developmental ages (3, 12, and 24 m) pulmonary emphysema and vascular remodeling were investigated in lungs of p66shc^–/– and wild-type (WT) mice in vivo by lung function, hemodynamics, and echocardiography, as well as by histological determination of the mean linear intercept, alveolar number, the degree of vascular muscularization, and by casting/µCT of pulmonary vessels. Lung cellular senescence was investigated by β-galactosidase staining and by the expression of the secretory-associated senescence phenotype (SASP). Results showed that in 24-months-old WT mice, the alveolar number was decreased compared to young mice, while the degree of muscularization of small pulmonary vessels was not altered. P66shc^–/– mice showed an accelerated decrease of the alveolar number compared to WT mice of the respective age even though lung function was not different. Despite similar right ventricular systolic pressure in p66shc^–/– and in WT mice, p66shc^–/– exhibited increased vascular muscularization and decreased vascular branching compared to 12-month-old WT mice. Furthermore, p66shc^–/– showed increased p21 expression at early age (3 m), increased accumulation of senescent cells at 12 months old, and higher SASP expression. In conclusion, the protein p66shc is necessary to maintain physiologic function of the lung. Deletion of p66shc has deleterious effects on the lung leading to accumulation of senescent cells, decrease of alveolar number, and pulmonary vascular remodeling.

Pharmacokinetic (PK) performance of LIQ861 and evaluation of comparative bioavailability with Tyvaso® in healthy subjects

Robert F. Roscigno, Toby Vaughn, Thomas Hunt, Ed Parsley, Mike Eldon and Lewis J. Rubin

Liquidia Technologies, Morrisville, NC, USA; PPD, Austin, TX, USA; Columbia University College of Physicians and Surgeons, New York, NY, USA

LIQ861 (861), a novel dry powder formulation of Treprostinil (TRE) is designed to enhance deep-lung delivery and achieve higher tolerated dose levels than current inhaled pulmonary arterial hypertension (PAH) therapies. We previously reported results from LTI-101, a placebo-controlled, double-blind, randomized, single-center study that evaluated the ascending single-dose pharmacokinetics (PK) of LIQ861 in healthy subjects. LTI-102 was a randomized, open-label, crossover study comparing single doses of LIQ861 and Tyvaso to determine comparative bioavailability. Twenty-four subjects were randomized to one of three treatment sequences (LIQ861/LIQ861, Tyvaso/LIQ861, LIQ861/Tyvaso). Sequence 1 assessed the reproducibility of LIQ861 dosing and systemic levels of treprostinil utilizing the RS00 Dry Powder Inhaler (Plastiape, Osagno It.). Sequences 2 and 3 evaluated the rate and extent of treprostinil exposure after LIQ861 administration (nominal 75 mcg dose/79.5 mcg capsule strength) compared to nine breaths (approximately 54 mcg). Tyvaso was administered as per US PI¹, utilizing the TD-300 nebulizer. The LTI-101 study demonstrated that treprostinil exposure from LIQ861 increased proportionally across all doses studied (25–150 mcg). The PK profile of treprostinil administered as LIQ861 was consistent with prior Tyvaso^® studies). All LIQ861 doses were generally well tolerated, and no deaths, serious AEs, or dose-limiting toxicities reported. In LTI-102, treatment emergent AEs related to LIQ861 and Tyvaso were mild to moderate in intensity and consistent with known prostanoid effects. In sequence 1, mean LIQ861 Cmax and AUC values were comparable, confirming reproducible dose delivery. In sequences 2 and 3, T_max and exposure parameters C_max, AUClast, and AUCinf were similar with slightly lower values for 861 than Tyvaso. C_max and AUC geometric mean ratios indicate treprostinil systemic exposure (dose delivered to the lung) after LIQ861 administration is > 90% of Tyvaso (Fig. 1). In conclusion, study LTI-102 demonstrates that LIQ861 and Tyvaso have comparable Treprostinil systemic exposures, justifying reliance on FDA’s previous finding of safety and effectiveness for Tyvaso.

Fig. 1.

Boxplots of observed PK parameters following administration of Tyvaso and LIQ861 to healthy subjects (n = 8) in study in LTI-102, Part 3.

Increased susceptibility to gammaherpesvirus-induced lung fibrosis of transgenic mice with conditional overexpression of the ER stress-factor chop in alveolar epithelium

Martina Korfei, Mohamed Smaida, Oleksiy Klymenko, Clemens Ruppert, Ingrid Henneke, Martin Huehn, Poornima Mahavadi, Susanne Herold, Werner Seeger, Heiko Adler and Andreas Guenther

Universities of Giessen and Marburg Lung Center (UGMLC), Biomedical Research Center Seltersberg (BFS), Justus-Liebig-University Giessen, Giessen, Germany; UGMLC, Department of Internal Medicine, Medical Clinic II, Giessen, Germany; German Center for Lung Research (DZL), Grosshansdorf, Germany; Department of Lung Development and Remodeling, Max-Planck-Institute for Heart and Lung Research, Bad Nauheim, Germany; Research Unit Lung Repair and Regeneration, Helmholtz Zentrum München – German Research Center for Environmental Health (GmbH), Munich, Germany; Agaplesion Lung Clinic Waldhof Elgershausen, Greifenstein, Germany; European IPF Network and European IPF Registry

Idiopathic pulmonary fibrosis (IPF) is an age-related interstitial lung disease with fatal outcome. The main characteristic of IPF is ER-stress and apoptosis in type-II alveolar epithelial cells (AECII), which in consequence cause chronic epithelial injury and progressive lung fibrosis. We developed double transgenic mice expressing the proapoptotic ER-stress-factor Chop exclusively in AECII by using the Tetracycline-On-system (SP-C rtTA/tetO7-Chop). In response to 28 days doxycycline-feeding (Dox+), nuclear Chop-overexpression in AECII resulted in AECII apoptosis, but not in development of lung fibrosis. We thus suggested that the extent of AECII apoptosis in the ‘Chop-mice’ was not sufficient to cause lung fibrosis, and that ‘second hits’ such as age, (herpes)virus-infection, ROS-exposure, etc. may be required for ‘full-blown’ AECII-ER-stress and ‘high-level’ AECII apoptosis. Indeed, several studies have suggested that herpesviruses may play a role in IPF-pathogenesis. We therefore infected Chop transgenic mice after 28 days transgene-induction (Dox+), as well as without transgene induction (Dox–), intranasally with murine gammaherpesvirus-68 (MHV68). At 15 days post infection, mice were sacrificed and analysed. Lungs of MHV68-infected (Dox+) Chop-mice, but not of infected (Dox–) Chop-mice or uninfected (Dox+) and (Dox–) Chop-mice, evidently showed thickening of alveolar septae and fibrotic remodelling. In addition, ER-stress and strong apoptosis-induction (caspase-9/-3-activation, PARP1-cleavage) was observed in lungs of both MHV68-infected (Dox+) and (Dox–) Chop-mice versus controls, presumably due to virus-induced lung injury, but was more pronounced in the infected (Dox+) Chop-mice. Interestingly, Chop was not (endogenously) induced in (Dox–) Chop-mice in response to MHV68-infection, despite evident virus-induced ER-stress! Furthermore, MHV68-infected (Dox+) and (Dox–) Chop-mice indicated upregulation of profibrotic protein-expression (Mmp2, Pai-1, p-Smad3) versus controls, but was ‘biggest’ in the infected (Dox+) Chop-mice. We conclude that our transgenic Chop-mouse model resembles human IPF, since IPF patients have been defined as (genetically) susceptible individuals prone to develop disease with second hits.

Dicarbonyl stress in right ventricular dysfunction in pulmonary arterial hypertension

Sasha Z. Prisco, Lynn Hartweck, Megan Eklund, Lauren Rose, Thenappan Thenappan and Kurt Prins

Cardiovascular Division, University of Minnesota, Minneapolis, MN, USA

Right ventricular dysfunction (RVD) is the leading cause of mortality in pulmonary arterial hypertension (PAH), but the molecular mediators of RVD are incompletely understood. RV glucose uptake is strongly and inversely proportional to RV function in PAH patients, which is believed to be due to metabolic derangements including increased glycolytic flux due to mitochondrial dysfunction. Methylglyoxal (MG) is a highly reactive dicarbonyl compound, which can post-translationally modify protein function. MG is formed during glycolysis and approximately 1% of glucose metabolized through glycolysis is converted to MG. The pathway that regulates MG levels uses glutathione and two key enzymes: glyoxalase (Glo) I and II. Finally, DJ-1 is a deglycase enzyme that can reverse protein methylglyoxation. Regulation of protein-MG, the Glo system, and DJ-1 in RVD are unexplored. We analyzed protein-MG, Glo-1/2, and DJ-1 expression in RV and LV extracts from control, monocrotaline (MCT), and pulmonary artery banded (PAB) rats. We analyzed the effects of protein-MG on mitochondrial function in H9c2 cardiomyocytes using Seahorse analysis. Finally, we examined the link between protein-MG and RV function in PAH patients using hemoglobin-A1C as a measure of protein-MG. Results showed that in MCT RV extracts, there was an increase in protein-MG (3.2 ± 0.9-fold), a 48 ± 7% reduction in Glo-1 with slightly increased levels of Glo-2 (1.4 ± 0.1-fold), and DJ-1 (1.5 ± 0.4-fold). In PAB rats, protein-MG (1.4 ± 0.3-fold) was not as drastically elevated, Glo-1 was slightly increased (1.7 ± 0.3-fold), Glo-2 slightly increased (1.6 ± 0.2-fold change), while DJ-1 was markedly elevated (2.3 ± 0.3-fold). In H9c2 cardiomyocytes, MG induced protein-MG and impaired mitochondrial function as quantified by maximal oxygen consumption. Finally, in PAH patients, increased hemoglobin glycation levels were inversely associated with RV contractility. In conclusion, increased protein-MG is associated with more severe RV dysfunction in pre-clinical RV pressure overload and impaired mitochondrial function in vitro. These results suggest excess protein-MG causes metabolic derangements which promotes RVD.

Interleukin-6 directly dysregulates the microtubule-junctophilin-2-T-tubule pathway to exacerbate right ventricular dysfunction in pulmonary arterial hypertension

Sasha Z. Prisco, Lauren Rose, Thenappan Thenappan and Kurt W. Prins

Cardiovascular Division, University of Minnesota, Minneapolis, MN, USA

Pulmonary arterial hypertension (PAH) is a progressive and lethal pulmonary arterial vasculopathy that causes right ventricle dysfunction (RVD). We recently demonstrated that microtubule-mediated junctophilin-2 (MT-JPH2) pathway downregulation promotes t-tubule disruption and RVD; however, the upstream regulators of this pathway are unknown. Interleukin-6 (IL-6), an inflammatory cytokine, promotes microtubule remodelling in isolated rat neonatal cardiomyocytes via its downstream signalling protein signal transducer and activator of transcription 3. However, the relationship between IL-6 and the MT-JPH2 pathway in RVD due to PAH is unknown. Immunoblots of RV extracts from control, monocrotaline (MCT) and MCT rats treated with an IL-6 antagonist were probed for the IL-6 and the MT-JPH2 pathways. Ventricular cardiomyocytes were isolated and cultured in the presence of IL-6, and confocal microscopy was used to examine microtubule remodelling, JPH2 localization and t-tubule organization. Echocardiography quantified RV function. Serum levels of IL-6 and the relationship to RVD in PAH patients (n = 73) were examined. Results showed that western blot analysis revealed activation of IL-6 signalling, microtubule remodelling and JPH2 downregulation in the RV of MCT rats. Culture with IL-6 induced MT remodelling, JPH2 mislocalization and pathological t-tubule remodelling in isolated cardiomyocytes. MCT rats treated with an IL-6 antagonist (two weeks after MCT injection) had reduced RV IL-6 signalling, increased JPH2 expression, normalization of t-tubule architecture, improved RV function, blunted RV hypertrophy and improved survival compared to MCT rats despite no differences in pulmonary artery acceleration time. PAH patients with higher IL-6 levels had higher N-terminal pro-brain natriuretic peptide and lower RV fractional area change despite no differences in mean pulmonary arterial pressure, pulmonary vascular resistance and pulmonary arterial compliance. In conclusion, IL-6 directly dysregulates the MT-JPH2 pathway. Inhibition of IL-6 signalling increases JPH2 expression, corrects t-tubule architecture, enhances RV function and improves survival in MCT PAH.

Surviving inhibition as a potential target for pulmonary arterial hypertention

I. Blanco, M. Marquina, O. Tura-Ceide, V.I. Peinado and J.A. Barberà

Department of Pulmonary Medicine, Hospital Clínic-University of Barcelona, Barcelona, Spain; Institut d’Investigacions Biomèdiques August Pi i Sunyer, Barcelona, Spain; Centro de Investigación Biomédica en Red de Enfermedades Respiratorias, Madrid, Spain

Imbalance between cell proliferation and apoptosis may underlie the development of pulmonary arterial hypertension (PAH). Survivin is a member of the apoptosis inhibitor gene family involved in cell proliferation. This study aimed to explore if the expression of survivin is increased in a mice model of PAH. We also investigated the effects of the survivin inhibitor (YM155) in the mice model. C57/BL6 mice were exposed to hypoxia and SU5416 (injected s.c. weekly) for three weeks. YM155, a suppressor of survivin expression, was also administered s.c. during seven days, the third week after hypoxia and SU5416 injection. We measured right ventricular systolic pressure (RVSP) and right ventricle (RV) hypertrophy by the Fulton index. Vascular remodeling and survivin expression were assessed in lung sections by immunohistochemistry for smooth muscle actin (SMA) and survivin, respectively. Survivin gene and protein expression were evaluated by qPCR and western blot, respectively. Results showed that compared with controls, SU5416 + hypoxia-treated mice showed a significant increase in RVSP (46 ± 15 vs. 18 ± 5 mmHg; p < 0.0001), RV hypertrophy (RV/LV + septum ratio, 0.37 ± 0.07 vs. 0.29 ± 0.02; p = 0.0005), vascular remodeling (64.1% vs. 43.6% SMA⁺ vessels/N° vessels⁺; p = 0.002), and an increased expression of survivin in intrapulmonary arteries (23.9% vs. 8.5% survivin⁺ vessels/N° vessels⁺; p = 0.0002). Survivin gene and protein expressions were twice higher in SU5416 + hypoxia mice compared to controls (p = 0.01). Treatment with YM155 reduced RVSP (46 ± 15 in sugen +hypoxia mice vs. 24 ± 5 in sugen + hypoxia + YM155 mice; p < 0.0001), right ventricular hypertrophy (0.38 ± 0.04 vs. 0.32 ± 0.14; p = 0.06), vascular remodeling, and protein–gene survivin expression (that was twice higher in mice exposed to hypoxia and SU5416 compared with mice exposed to SU5416 + hypoxia + YM155 (p = 0.003)). Survivin expression is increased in the SU5416 + hypoxia experimental mice model of PAH and concomitant treatment with YM155 prevents the development of PAH. In conclusion, these results suggest that survivin is involved in the pathogenesis of PAH and might represent a potential therapeutic target (This study was supported by FIS (PI17/1515) and SOCAP).

Modelling vascular responses in pulmonary arterial hypertension in a microfluidic pulmonary artery-on-a-chip platform

Alexander J. Ainscough, Joshua B. Edel and Beata Wojciak-Stothard

Department of Medicine, Imperial College London, London, UK; Department of Chemistry, Imperial College London, London, UK

Pulmonary arterial hypertension (PAH) is a debilitating disease for which there is currently no cure. Inhibition of bone morphogenetic protein receptor 2 (BMPR2) contributes to endothelial dysfunction and vascular remodelling in PAH. At present, no single animal model of PAH fully reflects human disease. To address this issue, we have created a microfluidic model of the human pulmonary vascular wall, the pulmonary artery-on-a-chip, where human pulmonary artery endothelial cell (HPAEC) and human pulmonary arterial smooth muscle cell (HPASMC) are co-cultured under physiological haemodynamic conditions in distinct microfluidic chambers corresponding to the size of the human peripheral lung arterioles affected by the disease. Primary HPAEC were transduced with adenoviruses containing short hairpin RNA against BMPR2. Blood-derived endothelial cells (BOECs) were obtained from healthy controls and PAH patients with BMPR2 haploinsufficiency. Endothelial permeability measurements were conducted using 40kDa FITC-Dextran perfused over the endothelial layer. Proliferation of endothelial and smooth muscle cells was assessed through fluorescent EdU staining. Cell alignment was studied with fluorescent confocal microscopy and ImageJ. Results showed that HPAEC exhibited preferential alignment to the direction of shear stress, whilst HPASMC exhibited perpendicular alignment to the direction of flow, resembling the orientation of arterial cells in vivo. Endothelial cells grown in the chip under physiological flow conditions showed significantly enhanced barrier function compared with the cells grown in static conditions and showed an increased sensitivity to stimulation with thrombin. Endothelial BMPR2 knockdown reduced endothelial cell adaptation to flow, and increased proliferation of HPASMCs, likely to reflect cell responses in the remodelled PAH lung. BOECs from PAH patients displayed abnormal alignment and proliferation under flow, compared with healthy controls. In conclusion, the pulmonary artery-on-a-chip displays arteriole-like features, recapitulates aspects of vascular dysfunction in PAH and therefore can serve as a tool for modelling changes seen in PAH. This approach is a step towards development of a novel precision medicine platform for patients with PAH and other cardiovascular diseases.

Relation of syncope to a baseline risk status, pulmonary artery obstructive burden and mortality in acute pulmonary embolism

Berhan Keskin, Ozgur Y. Akbal, Seda Tanyeri, Aykun Hakgor, Hacer C. Tokgoz, Ali Karagoz, Cem Dogan, Zubeyde Bayram, Seyhmus Kulahcioglu, Dogancan Ceneli, Ismail Balaban, Rezzan D. Acar, Ibrahim H. Tanboga, Nihal Ozdemir and C Kaymaz

Kartal Kosuyolu Heart Education and Research Hospital, University of Health Sciences, Istanbul, Turkey; Department of Biostatistics, Medical School, Ataturk University, Erzurum, Turkey; Department of Cardiology, Hisar Intercontinental Hospital, Nisantası University, Istanbul, Turkey; FACC Kartal Koşuyolu Yüksek İhtisas Eğitim ve Araştırma Hastanesi, İstanbul, Turkey

Although, pulmonary embolism (PE) has been reported up to 17% in patients (pts) admitted to emergency room with syncope, clinical impact of syncope in PE remains to be clarified. We aimed to evaluate clinical, echocardiographic, CT angiographic (CTA) correlates of syncope and its impact on short- and long-term mortality (LTM) in PE pts. Our study is based on retrospective analysis of 610 pts with PE (female 58%, median age 65.25 years) from 2011 to 2019, and pts were classified by the presence of syncope (S+, S–) to compare clinical parameters including systolic blood pressure (SBP), heart rate (HR), oxygen saturation (SPO₂), PESI score, echo parameters including TAPSE, systolic pulmonary artery (PA) pressure (SPAP), CTA measures including Qanadli score (QS), right ventricle/left ventricular diameter ratio (RV/LVr) and short and LTM, bleeding events. Results showed that overall 181 (29.7%) pts were S+. Demographic and baseline characteristics were comparable between two groups. Treatment choices were heparin, systemic-tPA (stPA) and catheter-directed-treatment in 29.8, 26.9, and 43.2% pts, respectively. The S+ pts had lower SBP (121; 110–135 vs. 112; 91–130), SPO₂ (90; 87–93 vs. 88; 85–92), and TAPSE (1.9; 1.6–2.2 vs. 1.7; 1-4-2) and higher HR (104; 91–115 vs. 113; 100–122), SPAP (50; 40–60 vs. 55; 45–62.5), PESI score (96; 76–119 vs. 120; 95–149), QS (20; 14, 75–25 vs. 24; 20–29), and RV/LVr (1, 11; 1–1.25 vs. 1.25; 1.13–1.41), respectively (p < 0.001 for all). SBP, HR, TAPSE, SPAP, QS, and RV/LVr were similar between groups after treatment (p > 0.05). stPA was used more in S+ pts (21.9% vs. 38.7%, p < 0.001). S+ group had a higher in-hospital M (6.1% vs. 12.7%, p = 0.009) while major bleeding was comparable between two groups (4.7% vs. 8.8%, p = 0.070), whereas minor bleeding was more in S+ (3% vs. 11%, p < 0.001). However, LTM (20.3% vs. 24.3%, p = 0.159) was comparable between groups during median 605 days (177–970) of follow-up. In conclusion, in our PE population, recent syncope related to a higher baseline risk status, more severe PA obstructive burden and RV dysfunction, and increased stPA utilization. Syncope seems to relate with higher in-hospital mortality, but not LTM.

Comparison of baseline and post-treatment measures in acute pulmonary embolism patients categorized by four different treatment modalities

Hakgor Aykun, A. Hakgor, Ozgur Y. Akbal, O.Y. Akbal, Berhan Keskin, B. Keskin, Seda Tanyeri, S. Tanyeri, Ali Karagoz, A. Karagoz, Hacer C. Tokgoz, H.C. Tokgoz, Cem Dogan, C. Dogan, Zubeyde Bayram, Z. Bayram, Seyhmus Kulahcioglu, S. Kulahcioglu, Sevim Turkday, S. Turkday, Rezzan D. Acar, Ibrahim H. Tanboga, I.H. Tanboga and Nihal Ozdemir

Kartal Kosuyolu Heart Education and Research Hospital, University of Health Sciences, Istanbul, Turkey; Department of Biostatistics, Medical School, Ataturk University, Erzurum, Turkey; Department of Cardiology, Hisar Intercontinental Hospital, Nisantası University, Istanbul, Turkey; Kartal Koşuyolu Yüksek İhtisas Eğitim ve Araştırma Hastanesi, İstanbul, Turkey

In this single-centre study based on retrospective analysis of six-year experience on acute pulmonary embolism (PE), we aimed to evaluate baseline characteristics, risk status and clinical outcome measures in patients (pts) who underwent different treatment modalities. Study population comprised 535 pts (female 309, age 62.2 ± 16.7 years) with PE at low, intermediate-low, intermediate-high or high-risk status. The systematic work-up including multidetector computed tomography, Echo, biomarkers, PE severity indexes and Qanadli score (QS). Four different treatment modalities were intravenous-heparin followed by low-molecular-weight heparin in 201 (37.5%), ultrasound-facilitated thrombolysis (UFT) in 207 (38.7%), rheolytic thrombectomy (RT) in 41 (7.6%), and systemic tissue-type plasminogen activator (st-PA) infusion in 86 (16.2%) pts. Results showed that age, gender and symptom-to-treatment interval were comparable between four treatment cohorts (p > 0.05 for all) while malignancy was more frequent in RT group (p = 0.026). On admission, vital signs, PE severity indexes and risk status were significantly unfavourable in pts who underwent st-PA treatment (p < 0.001 for all) while right/left ventricle diameter ratio (RV/LV), QS, PA systolic pressure and RV systolic functions were best in i.v.-heparin group (p < 0.05). Mean t-PA dosage and infusion duration in UFT and st-PA groups were 35.6 ± 13.5 and 59 ± 29.5 mg, and 23.8 ± 6.8 and 6.2 ± 4.9 h, respectively. Intra-PA t-PA bolus was needed in 11 pts (mean 15.7 ± 4.9 mg) treated with RT. Major bleeding rates were 11.6%, 9.8%, 7.2% and 2% in st-PA, RT, UFT and heparin groups, respectively. Minor bleeding was most frequent in UFT-arm (11.1%) and was usually related with access site. In-hospital and long-term mortality were 6% and 17.4%, 6.3% and 13.5%, 9.8% and 22%, 13.9% and 24.4% in heparin, UFT, RT and st-PA groups, respectively, that were comparable between four treatment groups (p = 0.092 and p = 0.177). In conclusion, our retrospective data seem to be consistent with evidence-based risk stratification followed by individualized treatment in acute PE. UFT, RT and st-PA were more frequently utilized in IHR and HR at the expense of increased major and minor bleeding risk.

Whole genome sequencing and integrative genomics analyses to identify genetic determinants of pulmonary vascular remodeling in COPD

Adel Boueiz, Wonji Kim, Sool Lee, Michael Wells, Raul S.J. Estepar, John E. Hokanson, George R. Washko, Peter J. Castaldi, Michael H. Cho and Edwin K. Silverman

for the COPDGene and TOPMed investigators

Channing Division of Network Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA; Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA; Division of Pulmonary, Allergy and Critical Care Medicine, University of Alabama at Birmingham, Birmingham, AL, USA; UAB Lung Health Center, University of Alabama at Birmingham, Birmingham, AL, USA; Department of Radiology, Surgical Planning Laboratory, Laboratory of Mathematics in Imaging, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA; Department of Epidemiology, University of Colorado, Denver, CO, USA; General Medicine and Primary Care, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

Mechanisms underpinning pulmonary vascular disease in COPD have not yet been well-elucidated. Pulmonary artery enlargement (PAE), measured by CT as PA/A ratio of the diameter of the pulmonary artery to that of the aorta > 1, correlates with pulmonary hypertension by right heart catheterization and is associated with worse clinical outcomes. Genome-wide association studies previously identified multiple associations with PAE, but these studies lacked comprehensive coverage of genetic variants and the biological functions of the associated variants are unknown. To identify novel genetic determinants for PAE and characterize the functions of PAE-associated variants, we performed a whole-genome sequencing (WGS) analysis and integrated the results with publicly available epigenomic data. A total of 5131 subjects with available WGS and PA/A data in the COPDGene non-Hispanic-Whites, COPDGene African-Americans, and ECLIPSE studies were analyzed. PA/A (> or ≤1) was tested for genetic associations using variants with minor allele frequency (MAF) > 0.01% in the TOPMed Freeze8 data, and adjusting for age, sex, smoking, and genetic ancestry. Separate analyses in each population were followed by a meta-analysis. We performed single-variant, gene-based, and pathway analyses. We then quantified the enrichment of PAE-WGS regions in DNase I peaks from ENCODE and Roadmap cell types. We identified two loci associated with PAE at genome-wide significance: one previously reported (15q31 near IREB2) and one new (12p12 near LMO3) risk loci. Additional novel loci approaching genome-wide significance included regions near FREM2, CHEK2, and ZNF516. Top signals were enriched for several cell types including BMP4-derived mesendoderm cells and fibroblasts. Gene-based and pathway analyses highlighted potentially relevant biological mechanisms, including immune response and cytoskeleton remodeling (FDR 10%). This study leveraged imaging, genomics, epigenomics, and advanced bioinformatics tools and provided insights into new putative genetic determinants of pulmonary vascular remodeling in COPD. Additional analyses are needed to replicate these findings and understand their consequences on downstream processes. (This study was supported by NHLBI K08HL141601, R01HL116931, R01HL124233, R01HL126596, R01HL116473, U01HL089897, and U01 HL089856. The COPDGene study (NCT00608764) is also supported by the COPD Foundation through contributions made to an Industry Advisory Committee comprised of AstraZeneca, Boehringer-Ingelheim, Genentech, GlaxoSmithKline, Novartis, and Sunovion).

AFR implantation can provide symptom relief in PAH patients

Ahmad Amin

Department of Heart Failure & Transplantation, Rajaee Cardiovascular, Medical & Research Center, Tehran, Iran

Pulmonary arterial hypertension (PAH) is a progressive disease with a high rate of morbidity and mortality. Despite significant advances in pharmacological therapies, many patients will continue to deteriorate with progressive symptoms of right ventricular failure. Creating a right to left shunt through the interatrial septum can decompress right-sided chambers, improve left ventricular filling, and help maintain cardiac output, providing symptom relief and preventing syncope. Atrial flow regulator (AFR) device can maintain a permanent shunt with a predetermined diameter and help hemodynamics in PAH patients. Eight PAH patients (mean age: 35.6 years) with recurrent syncope and right-sided heart failure despite pharmacological treatment were enrolled and AFR implanted under fluoroscopy with help of transesophageal echocardiography. Patients were followed for six months. One patient deteriorated after the procedure and underwent VA extracorporeal membrane oxygenation (ECMO) implantation and lung transplantation. AFR was associated with stopping syncope, improving 6MWT (302 ± 33 to 380 ± 32 m), and improving cardiac index (2.1 ± 0.43 to 2.7 ± 0.5) which maintained to six months. In conclusion, AFR implantation can provide symptom relief in PAH patients with recurrent syncope and RV failure. It maintained patency during follow-up and improved symptoms, 6-MWD, and cardiac index.

Pregnancy and pulmonary arterial hypertension—maternal and fetal outcomes and N terminal pro brain natriuretic peptide trends from a tertiary care centre with dedicated pulmonary hypertension clinic

Shine Kumar, Cini S. Prasad, Sudha Sumathi, Radhamony Kunjukutty, Nitu Puthenveettil, Amitabh C. Sen, Jeya B. Sivabalakrishnan and Krishna Kumar

Departments of Pediatric Cardiology, Obstetrics and Gynecology, Anesthesia, AIMS, Amrita Vishwa Vidyapeetham, Kerala, India

Pregnancy associated with pulmonary arterial hypertension (PAH) poses significant maternal and fetal mortality and morbidity risks. N terminal pro brain natriuretic peptide (NT-proBNP) trends are undescribed in this subset. Aim of this study was to describe maternal and fetal outcomes and NT-proBNP trends in pregnancy associated with PAH. We analyzed prospectively collected data of maternal and fetal outcomes of pregnancy associated with PAH (2008–2019). We identified 35 pregnancies for 22 women (mean age 27.9 ± 4.7 years, mean weight 50.6 ± 8.1 kg). The diagnoses were Eisenmenger syndrome (ES) (16, 72.7%), post-operative residual PAH (3, 13.5%), idiopathic PAH (2, 9.2%), and one (4.5%) had systemic lupus erythematosus. Twenty-three babies (65.7%) were born alive, gestational age of 35.1 ± 2.9 weeks, 47.8% at term, with a birth weight of 2.1 ± 0.8 kg. There was one neonatal death due to extreme prematurity, two still births, four medical termination of pregnancy, and five abortions. Eighteen babies (78.2%) had low birth weight (LBW) and four (17.4%) had intrauterine growth retardation. ES mothers had 60% premature delivery and 80% LBW babies. NT-proBNP levels were elevated in the initial 72 h post-delivery (median 138 pg/ml, range 64.5–955). A persistent rise beyond 72 h (median 686 pg/ml, range 341–6680) was associated with prolonged recovery postpartum (median post-delivery hospital stay 18 days, range 10–25) reflecting continued right ventricular (RV) stress and maladaptation. Overall mean hospital stay was 19 ± 9.8 days including mean intensive care unit stay of 4.8 ± 2.1 days with a single maternal mortality (4.5%). In conclusion, maternal and fetal outcomes of pregnancy associated with PAH are better but with concerning morbidity. Postpartum period remains the most vulnerable period. NT-proBNP trends may be useful to identify RV maladaptation and vulnerable subsets in postpartum period.

Bone morphogenic protein receptor 2 mutations in pulmonary hypertension in Indian population

Shine Kumar, Lalitha Biswas, Arul Arasan, Neethu Zachariah and Krishna Kumar

Departments of Pediatric Cardiology, Molecular Biology, Pulmonary Hypertension Clinic, Amrita Centre for Nano Sciences and Molecular Medicine, AIMS, Amrita Vishwa Vidyapeetham, Kerala, India

Bone Morphogenic Protein Receptor 2 (BMPR2) mutations are described in patients with pulmonary arterial hypertension (PAH). We aim to describe characteristics of BMPR2 mutations in Indian population. All patients with diagnosis of idiopathic (IPAH), hereditary (HPAH), associated with congenital heart disease (CHD) and residual PAH after CHD correction were included. BMPR2 mutation was analysed in peripheral vein blood sample by Sanger sequencing method. Study period was from January 2018 to October 2019. We analysed 46 patients (females 69.5% (32/46)), median age 26 years (0.7–45), including 16 (34.7%) children (< 18 years). The diagnosis were IPAH (33/46, 71.7%), HPAH (7/46, 15.2%,), PAH with associated CHD (5/46, 10.8%) and residual PAH (1/46, 2.2%). Thirteen patients (13/46, 28.2%) had BMPR2 mutation (69.2% females) with 43.7% (7/16) of children affected. Among four families with HPAH, two had mutations, both novel. Mutation was found in 21.2% (7/33) of IPAH with one new mutation, one patient (20%) with associated CHD and the only patient with residual PAH. Patients with mutations had varied age of presentation from 0.7 years to 38 years. All patients had severe PAH at presentation with only two patients (15.3%) responding well to combination therapy, one among them had uneventful pregnancy. A HPAH patient with novel mutation refractory to combination therapy had excellent response to imatinib therapy. Other family with HPAH had novel mutation with autosomal dominant inheritance and variable penetrance resulting in asymptomatic carrier state (two patients) to severe PAH. There were two mortality. In conclusion, in Indian population, patient with BMPR2 mutations may have predominantly early age of presentation with overall poor response to targeted therapy. Novel mutations were identified. HPAH-associated mutation was autosomal dominant with variable penetrance. Unique response to imatinib therapy in HPAH needs to be explored.

Evaluation of the change of six-minute walk test distance with conditioning

Kivrak Tarik, Polat İsmail, Karaca Ozkan and Karasu Mehdi

Department of Cardiology, Firat University, Elazig, Turkey

Pulmonary hypertension (PH) is a progressive disease characterized by an elevation of pulmonary artery pressure and pulmonary vascular resistance, leading to right ventricular failure and death. Exercise intolerance is the main characteristic of pulmonary arterial hypertension. The six-minute walking test (6MWT) is widely used in assessing exercise capacity of PH patients. Six-minute walking distance has been specified as the main clinical outcome in PH and has been used as the primary end-point in many studies conducted for new PH treatments. In addition, it has prognostic importance and is a good prognostic marker. However, there are many environmental and individual factors that affect the sensitivity and objectivity of the 6MWT, which has such an important role in PH. The aim of the study was to investigate whether the 6MWT distance may undergo significant changes with conditioning. Fifty patients with Group I and IV pulmonary hypertension who were admitted to outpatient clinic of Cardiology Department of Fırat University Medical Faculty (between August 2019 and September 2019) were included in the study. These patients were randomly divided into pairs, and first, one by one and then, two patients simultaneously were subjected to a 6MWT in accordance to the 2002 American Thoracic Society consensus. When the 6MWT results were examined, the mean distance of the 50 patients included in the study was 348.8 ± 97.7 m at the end of the first test, and 381.4 ± 95.3 m at the end of the second test. The second walking test showed a mean increase of 32.6 m or 9.3% than the first test. And this increase was found to be statistically significant (p < 0.01) (Table 1). In this study, it was observed that conditioning improved the performance of the patients and increased the walking distance. As a result of the study, it was demonstrated that conditioning can be used to increase the sensitivity and objectivity of the 6MWT.

Table 1.

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	First 6MWT distance	Second 6MWT distance	p Values
	Mean ± SD	Mean ± SD
Male	381.3 ± 112.1	405.1 ± 105.3	0.01
Female	340.7 ± 93.5	376.1 ± 93.1	<0.01
<50 age	400.2 ± 83.4	433.1 ± 80.4	<0.01
>50 age	301.4 ± 86.2	334.6 ± 83.7	<0.01
Total	348.8 ± 97.7	381.9 ± 96.3	<0.01

SD, standard deviation; 6MWT, 6 minute walking test.

Inducible nitric oxide synthase in myeloid cells drives smoke-induced pulmonary hypertension in mice

Marija Gredic, Simone Kraut, Cheng-Yu Wu, Stefan Hadzic, Oleg Pak, Rajkumar Savai, Baktybek Kojonazarov, Hossein A. Ghofrani, Werner Seeger, Friedrich Grimminger, Ralph T. Schermuly and Norbert Weissmann

Cardio-Pulmonary Institute, Universities of Giessen and Marburg Lung Center, Member of the German Center for Lung Research, Justus Liebig University, Giessen, Germany; Max Planck Institute for Heart and Lung Research, Member of the German Center for Lung Research, Bad Nauheim, Germany

Chronic obstructive pulmonary disease (COPD) is a progressive and incurable disorder that represents the third leading cause of death worldwide. Pathological changes in COPD include small airway remodeling, alveolar destruction, and activation of the innate immune system. Additionally, up to 90% of COPD patients develop at least mild pulmonary hypertension (PH), associated with increased morbidity and risk of exacerbations. We previously demonstrated the critical role of inducible nitric oxide synthase (iNOS) in development and reversal of PH and emphysema in smoke-exposed mice and showed that iNOS expression in bone marrow-derived cells drives pulmonary vascular remodeling, but not parenchymal destruction. In this study, we aimed to identify the iNOS-expressing cell type driving smoke-induced PH and decipher pro-proliferative pathways involved in this process. To address this question, we chronically exposed myeloid cell-specific iNOS knockout mice to smoke and monitored development of PH and emphysema. Additionally, we investigated pro-proliferative pathways in vitro, using co-cultures of bone marrow-derived macrophages and pulmonary artery smooth muscle cells (PASMC). Results showed that myeloid cell-specific knockout mice were protected against smoke-induced PH, as evident from hemodynamic measurements, echocardiography, and morphometric analysis of small pulmonary vessels. Confirming our previous findings, functional and morphometric analysis revealed emphysematous changes in both wild-type and myeloid cell-specific knockout animals exposed to smoke. Interestingly, myeloid cell-specific deletion of iNOS ameliorated the smoke-induced increase in expression of CD206, a marker of M2 polarization, on infiltrated macrophages. In vitro, pre-treatment of M2 macrophages with cigarette smoke extract potentiated the pro-proliferative signaling to PASMC in co-cultures, which was abolished by iNOS deletion in phagocytic cells. Our study demonstrates that iNOS deletion in myeloid cells protects mice against smoke-induced PH. It further implicates M2-polarized macrophages in smoke-induced pulmonary vascular remodeling and suggests that basal iNOS expression in this activation state can contribute to fine-tuning of the response to the environmental stimuli.

Cardiopulmonary exercise test in the evaluation of functional capacity in pulmonary hypertension

Maria L. Coronel, la Cal Teresa S. de, Angela F. Camacho, Fernando Arribas, Carmen P.O. Delgado, Maria T.V. Martin, Maria J. Lopez-Gude, Maria J.C. Ropero and Maria P.E. Subias

Hospital Universitario 12 de Octubre, Madrid, Spain

The assessment of functional capacity in pulmonary hypertension (PH) is essential in the initial workup and during follow-up. Exercise capacity can be evaluated by functional class (FC), six-minute walking test (6MWT), and cardiopulmonary exercise test (CPET). CPET parameters provide prognostic information and evaluate underlying pathophysiological mechanisms responsible of exercise limitation. In this context, idiopathic pulmonary arterial hypertension (IPAH) and chronic thromboembolic pulmonary hypertension (CTEPH) are two of the most prevalent groups of precapillary PH and the exercise limitation is the cardinal symptom. The objective of this study was to describe characteristics and exercise parameters in a cohort of patients with IPAH and CTEPH in a reference center in Spain. Until December 2018, 502 patients with CTEPH and 219 with IPAH were evaluated. For this analysis, we included 114 IPAH and 144 CTEPH with an initial workup assessment of functional capacity with CPET, 6MWT, and FC. Results showed that IPAH patients were younger and predominantly female. There were no significant differences in routine variables that assess functional capacity such as FC and 6MWT. IPAH showed worse hemodynamic parameters; however, the aerobic capacity and the parameters of ventilatory inefficiency were better than in CTEPH (Table 1). In conclusion, CPET discriminates more accurately exercise capacity than 6MWT in patients with PH. Aerobic capacity is reduced and ventilatory inefficiency is higher in CTEPH vs. IAPH despite having better hemodynamic parameters. These changes could be related to the deep alteration of pulmonary mismatch due to the presence of large areas not perfused in the CTEPH.

Table 1.

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Variable	IPAH (n = 114)	CTEPH (n = 144)	p Values
FC III–IV, n (%)	65 (57)	95 (66)	ns
6MWT (m)	447 ± 107	436 ± 110	ns
Pulmonary vascular resistance (UW)	13.7 ± 8.1	8.4 ± 4.3	<0.0001
Right atrial pressure (mmHg)	10.2 ± 14	7.7 ± 4	0.04
Cardiac output (l/min)	4.19 ± 1.4	4.57 ± 1.2	0.02
MVO2 (ml/kg/min)	16.8 ± 5.6	14.8 ± 7.5	0.02
EqCO2	35.5 ± 8.4	42.3 ± 7.9	<0.0001

IPAH, idiopathic pulmonary arterial hypertension; CTEPH, chronic thromboembolic pulmonary hypertension; FC, functional class; 6MWT, six-minute walking test.

Role of PARP1-PKM2/inflammation/oxidative DNA damage axis in the pathogenesis of right ventricular failure associated with pulmonary arterial hypertension

Tsukasa Shimauchi, Junichi Omura, Sandra Breuils-Bonnet, Valerie Nadeau, Roxane Paulin, Steeve Provencher, Olivier Boucherat and Sébastien Bonnet

Pulmonary Hypertension and Vascular Biology Research Group of Quebec Heart and Lung Institute, Laval University, Quebec, Canada

Right ventricular failure (RVF) is an independent poor prognostic factor for pulmonary arterial hypertension patients. Studies have revealed a metabolic shift from oxidative phosphorylation to glycolysis (Warburg effect) in compensated RV hypertrophy (cRVH). Nuclear pyruvate kinase muscle isozyme M2 (PKM2) promotes Warburg effect and PKM2/PKM1 ratio is established as a marker of glycolysis. Although the DNA damage response protein Poly ADP-ribose polymerase 1 (PARP1) was documented to induce PKM2 nuclear translocation and enhances Warburg effect in cancer, their role in RVF has never been studied. We hypothesized sustained PARP1 activation accounts for nuclear PKM2 localization, contributing to cardiomyocyte dysfunction and thus decompensated RV hypertrophy (dRVH). We found by Western blot that PKM2/PKM1 ratio was upregulated in cRVH (Cardiac index > 2.2, n = 14) and dRVH patients (died with RVF, n = 9), compared to control donors (n = 14), and correlated with RV hypertrophy and fibrosis. Similar findings were found in three PH rat models (monocrotaline, pulmonary artery banding, and Sugen–hypoxia). Interestingly, cardiac inflammation (NF-kappaB, IL-8, CD68), oxidative DNA damage (PARP1, MTH1, 8-oxodG), and apoptosis (TUNEL) were significantly increased in dRVH compared to cRVH despite similar PKM2/PKM1 ratios. Specifically, PARP1 expression inversely correlated with cardiac output and positively correlated with ANP, RV hypertrophy, and fibrosis; suggesting that persistent DNA damage repair by PARP1 promotes dRVH. Using neonatal rat RV cardiomyocytes, we confirmed DNA damage promotes nuclear expression of PARP1 and PKM2, leading to inflammation (NF-kappaB), oxidative DNA damage (8-OxoDG), and apoptosis (TUNEL). PARP1 inhibitor (ABT888) prevents nuclear retention of PKM2 and suppresses cardiomyocyte dysfunction (inflammation, oxidative DNA damage, apoptosis) (all p < 0.05). In conclusion and we demonstrated for the first time that PARP1-PKM2/inflammation/oxidative DNA damage axis is implicated in the transition from cRVH to dRVH and suppression of PKM2 nuclear function by PARP1 inhibitor may represent a new strategy to improve RV function.

Cor pulmonale in multi-facet miner lung disease: should we lower the standard echo thresholds?

Kemi Tibazarwa

LHC, Dar es Salaam, Tanzania

Lung disease has been well-documented in coal mines of the Western world and of South Africa. However, it remains that prevention is fundamental in avoiding complications, due to the difficulty in treatment, and the unpredictable responses to existing treatment modalities. In developing countries, diagnosis and management is even more challenging due to the difficulty in accessing expensive tests, frequently; as well as the expertise to articulate management flow. We describe the case of an overground-miner whose out-door exposure to mining dust none-the-less led to severe associated respiratory disease. We present a 45-year-old male, who, after several months of symptoms, was thought to have asthma; with the differential of chronic obstructive airway disease (COPD). Over the next two-year period, his symptoms would progress; responding poorly to treatment for common and atypical infective sequelae of obstructive airway disease. Sequential radiology suggested pneumoconiosis, with differential of silicosis, and interstitial lung disease. However, initial echo showed no significant right-sided disease. Twelve months later, the patient rapidly deteriorated into cor pulmonale; with pulmonary pressures still appearing only modestly raised. Despite treatment, he experienced several COPD exacerbations, and fluctuated in and out of cor pulmonale, even without COPD exacerbations; until it became almost refractory after another 12-month period. In all this time, pulmonary pressures never exceeded modest values, when clinically his right-heart failure had progressed. In conclusion, cor pulmonale resulting from mining-dust-related restrictive and obstructive lung disease is a severely debilitating condition. Although prevention of the underlying lung disease is most recommended, difficulties in diagnosis and management make this impractical. Cor pulmonale appears to hasten clinical deterioration, but inability to confirm echo criteria for right-heart disease can mislead clinicians away from the diagnosis. Applying revised echo criteria specific to these patients may be beneficial in improving symptoms, and perhaps outcome, by enabling stricter management earlier on.

Results of a screening program for PAH in HIV patients treated in hospitals in Argentina

E. Calegari, D. Litewka, T.R. Gomez, A.N. Atamañuk, D.R. Cuatz, V Fridman, V Giovini, N. Mastandrea, Sebastian Rivadeneira, D. Hoffmann, P. Rattagan and M. Payaslian

Htal Fernández, Htal Clinics, Buenos Aires, Argentina

Prevalence of pulmonary arterial hypertension (PAH) is 15–52 per 100,000. In HIV patients, it occurs in 1/200. Diagnosis appeared to be difficult. The VI World Symposium on Pulmonary Hypertension recommends the search for PAH in symptomatic or asymptomatic HIV patients with risk factors. The objective of this study was to describe the clinical and hemodynamic characteristics of patients with PAH/HIV detected in two hospitals in Buenos Aires, Argentina, at the time of diagnosis, and compare results with international registries. Cross-sectional description of 13 HIV patients diagnosed with PAH by CCD was performed. HIV patients with dyspnea or other guiding symptom and without other pathology that justify it referrals to the office of PH studied by echocardiography and CCD. Results are shown in Table 1.

In conclusion, patients showed clinical conditions indicating AIDS (Clinical Category C, CDC). At diagnosis time of PAH, 75% Q of the sample stayed with advanced disease but acceptable CD4 values. All were at intermediate risk of mortality for PAH. In spite of increased PVR and RV enlargement, preserved CI suggested a timely intervention.

Table 1.

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	n	Median	Q 1/3
CD4 (mm3)	13	320	280/376
FC (WHO)	13	3	2/3
6MWT (m)	13	300	210/320
NT-proBNP (pg/l)	12	375	83/717
Doppler PSAP (mmHg)	13	70	55/83
mPAP (mmHg)	13	43	35/55
PVR (Wood)	13	7.7	4.3/11
Wedge P (mmHg)	13	12	11/14
CI (L/min/m2)	13	2.7	2.3/3.9
	Htal.FernandezHtal. Clinicas	REVEAL	French registration	French HIV registration
n	13	51	42	35
Centers	2	55	17	14
n/centers	6.5	0.9	2.4	2.5
FC III / IV (%)	100		81
Doppler PSAP (mmHg)	43		49	46/30
PVR (Wood unit)	7.7		9.2	10/4
Wedge P (mmHg)	12		7	7/8
CI (L/min/m2)	2.7		2.7	3/3.6

FC, functional class; 6MWT, six-minute walking test; mPAP, mean pulmonary artery pressure; PVR, pulmonary vascular resistance; CI, cardiac index.

Proton MRI biomarkers of pulmonary arterial hypertension

Susan R. Hopkins, Ann R. Elliott, Rui C. Sá, Kevin J. Anderson, Abhilash K. Puliyakote, Beni Pazar, Nick H. Kim and G.K. Prisk

Department of Medicine, Radiology, University of California, San Diego, CA, USA

Functional imaging of regional pulmonary perfusion using arterial spin labeling proton MRI (ASL-FAIRER) has the potential to provide increased sensitivity to early pulmonary vascular disease because of the ability to detect locally impaired function that is obscured by otherwise normal lung. We hypothesized that pulmonary arterial hypertension (PAH, WHO group I) manifests as a progressive alteration in the pulmonary circulation with: increases in the volume of un-perfused regions as vessels succumb to remodeling (volume of absent perfusion (VAP) = % of lung ROI with signal characteristics indistinguishable from noise), increases in the spatial heterogeneity of perfusion (relative dispersion = SD/mean), and increases in the spatial heterogeneity of the fluctuations in perfusion over time (fluctuation dispersion, calculated as the SD of the change in perfusion from baseline in a time series) reflecting a failure of local pressure regulation. As a first step to developing early PAH biomarkers, we evaluated these metrics in patients with PAH (9 F, age = 51 ± 11 years, height = 161 ± 9 cm, weight = 62 ± 12 kg) and controls (7 F, 2 M, age = 40 ± 16 years, height = 165 ± 12 cm, weight = 72 ± 12 kg). ASL-FAIRER was used to measure regional pulmonary perfusion in a single sagittal slice in the right lung. Subjects were imaged supine in a 1.5 T GE MR scanner. There were no significant differences between PAH and controls for age (p = 0.11), height (p = 0.43), or weight (p = 0.08); however, FEV1%pred (p = 0.02) and FVC%pred (p = 0.01) were significantly reduced in PAH. VAP was significantly increased in PAH (9.1 ± 0.07% PAH, 2.8 ± 0.04% controls, p = 0.02) as was relative dispersion (1.28 ± 0.28 PAH, 0.93 ± 0.11 controls, p = 0.003) and fluctuation dispersion (0.38 ± 0.11 PAH, 0.17 ± 0.04 controls, p < 0.0001). Combined, the two spatial metrics, VAH and relative dispersion, perfectly distinguished the groups (area under the ROC curve = 1.0) as did the fluctuation dispersion (area under the ROC curve = 1.0). In conclusion, proton MRI perfusion biomarkers provide a high degree of sensitivity and specificity for PAH and may provide useful noninvasive screening tools. (This study was supported by NIH HL129990, HL122753, HL118539).

Mir-150-PTPMT1-cardiolipin signaling in pulmonary arterial hypertension

Giusy Russomanno, Kyeong B. Jo, Vahitha B. Abdul-Salam, Martin R. Wilkins and Beata Wojciak-Stothard

National Heart and Lung Institute, Imperial College London, London, UK

Endothelial dysfunction contributes to the development and progression of vascular pathology in pulmonary arterial hypertension (PAH). Circulating levels of miR-150 are reduced in PAH and act as an independent predictor of patient survival. Mechanistic links between changes in miR-150 levels and dysregulation of endothelial function in PAH are not fully understood. miR-150-induced changes in gene expression in human pulmonary artery endothelial cells (HPAECs) were analysed by RNA sequencing. Effects of miR-150 supplementation and endothelium-specific miR-150 knockdown were studied in Sugen/hypoxia mice, blood outgrowth endothelial cell (BOECs) from IPAH patients and HPAECs. Expression and localization of miR-150 and its key targets were studied by in situ hybridization and qPCR. Pulmonary endothelial and smooth muscle cell proliferation, inflammatory activation and mitochondrial function were also studied. Results showed that endothelial miR-150 levels were significantly reduced in lungs of PAH Sugen/hypoxia mice and BOECs from IPAH patients, compared with controls. DACC-mediated miR-150 delivery to the lung endothelium attenuated PH, while endothelium-specific knockdown of miR-150 aggravated the disease. RNAseq analysis revealed significant association of miR-150 targets with cell proliferation, inflammation and cardiolipin pathways. Overexpression of miR-150 and PTEN-like mitochondrial phosphatase (Ptpmt1), which is the top protein upregulated by miR-150, reduced hypoxia-induced cell proliferation, TNF-α-induced NFkB activation and improved mitochondrial function in HPAECs and BOECs from IPAH patients. Beneficial effects of miR-150 supplementation in vitro and in vivo were associated with increased PTPMT1 expression and PTPMT1-mediated biosynthesis of mitochondrial phospholipid cardiolipin. In conclusion, activation of miR-150-PTPMT1-cardiolipin pathway attenuates pulmonary endothelial damage induced by vascular stresses and may be considered as a potential therapeutic strategy in PAH.

Pulmonary arterial hypertension with below threshold pulmonary vascular resistance

Seshika Ratwatte, James Anderson, Geoffrey Strange, Carolyn Corrigan, Nicholas Collins, David S. Celermajer, Nathan Dwyer, John Feenstra, Dominic Keating, Eugene Kotlyar, Melanie Lavender, Helen Whitford, Ken Whyte, Trevor Williams, Jeremy Wrobel, Anne Keogh and Edmund M. Lau

on behalf of the PHSANZ Registry

Department of Cardiology, Concord Repatriation and General Hospital, Concord West, Australia; Royal Prince Alfred Hospital, University of Newcastle, New South Wales, Australia; Respiratory Department, Sunshine Coast Hospital, Queensland Australia; School of Medicine, University of Notre Dame, Fremantle, Australia; Heart Transplant Unit, St Vincent’s Hospital, Darlinghurst, Australia; Department of Cardiology, John Hunter Hospital, Newcastle, Australia; Sydney Medical School, University of Sydney, Sydney, Australia; Royal Prince Alfred Hospital, Camperdown, Australia; Department of Cardiology, Royal Hobart Hospital, Hobart, Australia; Queensland Lung Transplant Service, Prince Charles Hospital, Chermside, Australia; Department of Allergy, Immunology and Respiratory Medicine, The Alfred Hospital, Melbourne, Australia; Heart Transplant Unit, St Vincent’s Hospital, Darlinghurst, Australia; Advanced Lung Disease Unit, Fiona Stanley Hospital, Murdoch, Australia; Greenlane Clinical Centre, Auckland City Hospital, Auckland, New Zealand; Pulmonary Hypertension Society of Australia and New Zealand

Pulmonary vascular resistance (PVR) > 3 Wood Units (WU) is a criterion of the haemodynamic definition of pulmonary arterial hypertension (PAH). However, this conservative cut-off is not evidence based. Data are lacking on the natural history, response to therapy and survival of patients diagnosed with precapillary pulmonary hypertension with mild elevation of PVR. Using the Australian and New Zealand Pulmonary Hypertension Registry, we analysed outcomes of this unique group treated with PAH therapy between 2004 and 2017. Treatment for PAH could be prescribed solely on the basis of mean pulmonary arterial pressure (mPAP) and pulmonary artery wedge pressure (PAWP) in Australia. Thus, we studied patients with mPAP ≥25 mmHg, PAWP ≤15mmHg but PVR < 3WU. A total of 82 patients met the haemodynamic inclusion criteria (mean age 63 ± 11 years; 67 females). Underlying aetiologies included idiopathic disease (n = 39), connective tissue disease (n = 42) and HIV infection (n = 1). At diagnosis, mPAP was 27 mmHg (IQR: 25–30), PAWP 13 mmHg (IQR: 11–14) and PVR 2.2 WU (IQR: 1.9–2.7). Baseline 6MWD was 352 m (IQR: 280–416) and 77% were in NYHA 3 or 4 functional class. All patients were commenced on monotherapy with an endothelin receptor antagonist (n = 66) or phosphodiesterase-5 inhibitor (n = 16). At first re-evaluation (median five months; IQR: 4–12), 6MWD increased by 46 m (IQR: 7–96) and 35% demonstrated improvement in NYHA functional class. After a median follow-up of 65 months (IQR: 32–101), 18/82 (22.0%) had died, with estimated one-year and five-year survivals of 98% and 84%, respectively. Death attributed to PAH occurred in 6/18 (33.3%) of these patients. In conclusion, patients with precapillary PH and ‘borderline’ PVR have adverse outcomes. Such patients appear to respond to PAH therapy but this requires further study in randomised trials.

Rare variant association study of multiple pulmonary hypertension phenotypes using a Bayesian statistics framework

E.M. Swietlik, K.A. Lutz, D. Greene, M.W. Pauciulo, T. Tilly, N. Zhu, M. Cogliano, Carrie L. Welch, A.W. Coleman, Y. Shen, A. Swift, W.K. Chung, W.C. Nichols, N.W. Morrell and S. Gräf

University of Cambridge, Cambridge, UK; Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; Columbia University, New York, NY, USA; University of Sheffield, Sheffield, UK; NIHR BioResource – Rare Diseases Consortium, PAH National Cohort Study, US PAH Biobank

Precision medicine approaches require genotype–phenotype associations that have translational utility and hence can impact disease management and outcomes. To date, approximately one-quarter of patients with pulmonary arterial hypertension harbour rare mutations in disease-causing genes. We hypothesised that integrating deep phenotyping data with whole-genome sequencing data will reveal new rare disease-associated variants with smaller effect sizes. We analysed whole-genome sequencing data from 13,037 participants enrolled in the NHIR BioResource-Rare Diseases study, of which 1148 were recruited to the PAH domain. To test for genetic associations between genes and selected phenotypes of pulmonary hypertension (PH), we used a Bayesian model comparison method called BeviMed. By assigning phenotypic tags based on the current diagnostic classification of PH, stratification by age at diagnosis and transfer coefficient of carbon monoxide (KCO), we defined the groups for comparison. Results show that a strong association was identified between protein truncating variants (PTV) in KDR and significantly reduced KCO (PP = 0.989). KDR PTV carriers also presented significantly later in life (PP = 0.912). All patients harbouring PTV in KDR (n = 4) had mild parenchymal lung changes. One of the KDR subjects had a family history of PAH. KCO stratification also highlighted an association between IDH3G (n = 6) and moderately reduced KCO in patients with PAH (PP = 0.920). We identified four additional individuals with PTVs in KDR and three patients harbouring missense variants in IDH3G in the US PAH Biobank. We also confirmed associations between previously reported genes and PAH. In conclusion, deep phenotyping enabled patient stratification into subgroups with shared pathobiology and increased the power to detect new PAH risk genes namely KDR and IDH3G. These new genes further implicate central roles for the endothelium and alterations in mitochondrial respiration in the pathobiology of PAH.

Distal pulmonary artery malformations suggest poor prognosis in pulmonary arterial hypertension

Jun Wan, Yunxia Zhang, Huiwei Yang, Wanmu Xie, Bin Cao and Zhenguo Zhai

Department of Pulmonary and Critical Care Medicine, Center of Respiratory Medicine, China-Japan Friendship Hospital, Beijing, China; National Clinical Research Center for Respiratory Diseases, Beijing, China; Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China; Department of Respiratory Medicine, Capital Medical University, Beijing, China

The coexistence with distal pulmonary artery malformations (PAMs) is rare in pulmonary arterial hypertension (PAH) and challenged with complicated clinical situations. Here, we report two cases of PAH with distal PAM. Case 1: A 25-year-old male patient was diagnosed with PAH after a sudden syncope on 20 April 2017. His RHC data showed: mean pulmonary arterial pressure (mPAP): 49 mmHg, pulmonary artery wedge pressure (PAWP): 11 mmHg, pulmonary vascular resistance (PVR): 9.89 WU, cardiac output (CO): 3.84 l/min, and atrioventricular reentrant tachycardia (AVRT) (–). Computed tomography pulmonary angiogram (CTPA) found multiple distal PAM (pulmonary arteriovenous fistulae) with alveolar bleeding. He got poor response to tadalafil and ambrisentan, and had progressive dyspnea, intermittent hemoptysis, and chest pain. His second RHC (2019) showed worsened hemodynamics (mPAP: 69 mmHg, PAWP: 1 mmHg, PVR: 26.97 WU, and CO: 2.52 l/min). His gene analysis identified: BMPR2 gene c.1243G > A heterozygous missense mutation. He suffered from a fatal hemoptysis in 31 March 2019. Case 2: A female patient, 25 years old, started repeated hemoptysis from June 2013, but got PAH diagnosis in February 2019. Her RHC showed: mPAP: 78 mmHg, PAWP: 9 mmHg, PVR: 22.55 WU, CO: 3.06 l/min, and AVRT (–). CTPA found multiple PAM. Pulmonary arteriography showed multiple aneurysm. Gene detection identified BMPR2 gene c.1305_1307delTTTinsG heterozygous frameshift mutation. She got poor results from ambrisentan and treprostinil and had a fatal hemoptysis on 24 August 2019. In conclusion, PAH is featured with loss and obstructive remodeling of the pulmonary vascular bed, which usually leads to peripheral pulmonary vascular sparseness. While the distal PAM were found in both of these two patients, which were different from common cases of PAH. They shared the similar manifestation: young age, serve hemodynamic dysfunction, poor response to vasodilator (target treatments), refractory, and fatal hemoptysis. Gene sequencing identified BMPR2 gene mutation in these patients, which suggested possible pathogenesis for them. In conclusion, distal PAM related with fatal hemoptysis, which suggest poor prognosis in PAH patients.

Pediatric pulmonary hypertension at Children Hospital “Dr Ricardo Gutierrez”, Buenos Aires, Argentina

Mariana Cazalas, Inés Abella, María Sicurello, Alejandro Goldsman, Luis Trentacoste, Haydée Vazquez, María Grippo and Willy C. Parodi

Department of Cardiology, Ricardo Gutierrez Cildren’s Hospital, Buenos Aires, Argentina

Pulmonary hypertension is a rare but serious condition in pediatric patients caused by several etiologies. The objective of this study was to describe our population with pulmonary hypertension (PH). This is a retrospective and descriptive study; 60 patients (pts) with PH admitted to our hospital between August 2000 and August 2019 were included. Persistent PH of the newborn syndrome and Group3 of Nice 2018 Classification were excluded, except PH associated with developmental lung disorders. The analyzed variables were: gender, etiology, functional class (FC) at diagnosis, presentation symptom, therapeutic scheme, transition to adult care centers, transplant, and mortality. Gender: 53.33% female, but Idiopathic PH: 81.81% were female. Etiology According to Nice 2018 Classification: Group 1: 41 pts: 11 pts idiopathic PH, 1 pt familiar PH, 1 pt connective tissue disease, 24 pts congenital heart disease, 5 pts portal PH; Group 2: 1 pt: pulmonary vein stenosis; Group 3: 8 pts bronchopulmonary dysplasia; Group 4: 0 pt; Group 5: 9 pts: 1 pt pulmonary histiocytosis, 1 pt sickle cell anemia, 7 pts single ventricle. Eighteen percent of patients had genetic syndrome (100% Down syndrome)—FC at diagnosis: I (1 pt), II (22 pts), III (26 pts), IV (11 pts)—The most frequent presenting symptom at diagnosis was dyspnea, only 1 pt was asymptomatic. Syncope (7 pts): all of them with idiopathic PH. Transition to adult care centers was successful in eight patients. Transplant: three patients were listed. Only one underwent transplant others died in waiting list. Two patients are being evaluated. Mortality: 11 pts (18.64%)—therapeutic scheme: since 2007: PDE5i (18/60 pts = 28.33%); ERA (3/60 pts = 5%); Phosphodiesterase-5 inhibitors (PDE5i) and endothelin antagonist receptors (ERA) (20/60 pts = 33.3%); PDE5i + ERA + prostanoids (13/60 pts =21.66%); calcium channel blocker (2/60 pts = 3.33%); without treatment (4/60 pts = 6.66%). In conclusion, (1) functional class at diagnosis shows us a late diagnosis of PH; (2) The most frequent etiology was PH associated with congenital heart disease; and (3) combination therapy was the most frequent therapeutic scheme in the last 12 years.

NO-releasing organic nitrites in acute pulmonary hypertension: investigation of tolerance development in an experimental porcine model

C. Frostell, W. Gozdzik, S. Zielinski, P. Skrzypczak, K. Ratajczak, J. Albert and K.F. Nilsson

Department of Anesthesia and Intensive Care, Karolinska Institutet at Danderyd Hospital, Stockholm, Sweden; Department and Clinic of Surgery, Wroclaw Medical University, Wroclaw University of Environmental and Life Sciences, Wroclaw, Poland; Department of Cardiothoracic and Vascular Surgery, Örebro University, Örebro, Sweden

Organic nitrates, e.g. nitroglycerin, exhibit extensive tolerance development to itself and other organic nitrates (i.e. cross-tolerance) in long-term applications. The recently developed nitric oxide donor for intravenous administration, the organic mononitrites of 1,2-propanediol (PDNO), has been shown to have some selectivity for vasodilation in the pulmonary circulation in several relevant models of acute pulmonary hypertension. Interestingly, cross-tolerance in pulmonary and systemic vasodilation between nitroglycerin and PDNO did not develop in experimental acute pulmonary hypertension in pigs (Nilsson et al., 2018, Drug Des Devel Ther). The aim of the present study was to investigate if PDNO developed tolerance to itself in acute pulmonary hypertension induced by multiorgan failure. In anesthetized, mechanically ventilated, and instrumented pigs (n = 6), the effects of PDNO (15–60 nmol kg⁻¹ min⁻¹) on pulmonary and systemic arterial pressures and the concentration of nitric oxide in exhaled gas (chemiluminescence) were compared at baseline and after a combination of aortic cross-clamping (90 min) followed by reperfusion and a 10-h intravenous infusion of PDNO (45 nmol kg⁻¹ min⁻¹). Results showed that intravenous PDNO caused a similar increase of the concentration of nitric oxide in exhaled gas and decrease in systemic arterial pressure before and after the 10-h infusion of PDNO. At baseline, PDNO in increasing dose only had a slight lowering effect on the normal pulmonary arterial pressure, whereas it completely decreased the increased pulmonary arterial pressure after aortic cross clamping and reperfusion. In conclusion, intravenous PDNO was free of tolerance development to itself in a clinically relevant model of acute pulmonary hypertension. This is an advantageous characteristic of the substance compared to organic nitrates in clinical use.

Genome-wide association study of persistent pulmonary hypertension of the newborn in Thais

N. Nakwan, K. Singkhamanan, S. Mahasirimongkol, N. Satproedprai, T. Chaiyasung, P. Kunhapan and C. Charalsawadi

Faculty of Medicine, Department of Biomedical Sciences, Department of Pathology, Prince of Songkla University, Songkhla, Thailand; Division of Genomic Medicine and Innovation Support, Department of Medical Sciences, Medical Genetics Center, Ministry of Public Heath, Nonthaburi, Thailand

Genome-wide association study (GWAS) has not been reported for persistent pulmonary hypertension of the newborn (PPHN). Previous studies proposed several genetic risks that may explain the small proportion of the genetic contribution for PPHN. The objective of this study was to identify genetic variants associated with PPHN using a GWAS. This study was carried out on Thai subjects, including 45 PPHN patients and 294 normal subjects as a control group. Total of 659,184 SNPs were genotyped across the genome using Illumina Asian Screening Array-24 v1.0 BeadChip Array. We applied standard SNP quality-control filters to exclude SNPs with a low call rate (< 98%), a Hardy–Weinberg equilibrium (P < 1 × 10⁻⁸) for controls. A principal component analysis was performed to exclude genetic outliers and confirm the absence of significant genetic stratification between cases and controls. After quality control, we obtained 498,160 autosomal SNPs for the GWAS analysis. In this study, the genomic inflation factor, lambda, was 1.008 and the Manhattan plots are presented in Fig. 1. Two SNPs, rs140276032 (P = 5.04 × 10⁻⁹) noted to be in the intergenic region in proximity to genes for the CDC14C gene and rs149768622 (p = 3.40 × 10⁻⁸) located in the intron of WWC2 gene, achieved genome-wide significance (P < 1 × 10⁻⁸). Other top 10 suggestive SNPs (p < 1 × 10⁻⁵) were also identified. Of these, five (rs56074206, rs76080291, rs116280349, rs187729743, rs145552555) were located in the intron of five candidate genes (CIT, HDAC2, LOC100188947, LCORL, CYP39A1, respectively), and another five SNPs (rs375826735, rs143692554, rs140485153, rs141311709, rs10239513) were noted to be in intergenic regions. One SNPs (rs17034984) at the intron of the EPAS1 gene passed the Bonferroni threshold for significance was identified (P = 0.00014). In conclusion, this preliminary GWAS analysis showed that CDC14C and WWC2 might be associated with PPHN. In addition, our findings suggest that EPAS1 gene might be a key factor to contribute to the development of PPHN. However, a larger sample size is necessary to validate this association.

Fig. 1.

The Manhattan plot shows P-values for PPHN patients versus normal controls using the logistic regression analysis. X-axis represents the SNP markers in each chromosome and Y-axis shows negative logarithm of P-values. Total of 498,160 SNPs are presented. The highest P-value (P=5.04x10^- 9) was observed in the rs140276032 on the intergenic area in proximity to genes for CDC14C gene. Second high P-value (P=3.40 × 10^-8) was observed in the rs149768622 in intron of the WWC2 gene.

RV–PA coupling ratio using TAPSE/PAAT is not a reliable measure of severity of pediatric pulmonary hypertension

Pei-Ni Jone and CourtneyDunbar Cassidy

Department of Pediatric Cardiology, Children’s Hospital Colorado, University of Colorado, Aurora, CO, USA

Tricuspid valve annular systolic excursion (TAPSE)/pulmonary artery acceleration time (PAAT) has been suggested as an estimate of right ventricular (RV) to pulmonary artery (PA) coupling ratio in pulmonary hypertension (PH). The aim of this study is to evaluate TAPSE/PAAT ratio between normal controls and pediatric PH patients and to correlate this ratio with invasive hemodynamics to evaluate the severity of PH. Normal controls and PH patients underwent two-dimensional echocardiography from 2014 to 2016. PH patients underwent right heart catheterization within six months of echocardiograms. PAAT and TAPSE were measured with three cardiac cycles and averaged. Student t-tests of TAPSE/PAAT were performed between the groups. Correlations were performed with this ratio and invasive hemodynamics. Results showed that 40 controls were compared to 96 PH patients. There were no significant differences of this ratio between controls and PH patients (0.01 ± 0.005 vs. 0.01 ± 0.1, p = 0.53). There were statistically significant but weak correlations between this ratio to systolic pulmonary artery pressure (r = 0.44, p < 0.001), mean pulmonary artery pressure (r = 0.42, p < 0.001), pulmonary vascular resistance index (PVRi) (r = 0.38, p = 0.003), and PVRi/systemic vascular resistance index (r = 0.29, p = 0.006). In conclusion, TAPSE/PAAT cannot be used as a reliable measure of RV–PA coupling ratio as it is not statistically different between controls and PH patients. There is weak correlation between this ratio to invasive hemodynamics and thus cannot be used as a reliable measure of severity of PH.

Phenotypic characteristics according to genetic etiology

C. Pérez-Olivares, J. Tenorio, M.J. Cristo, V. Prudencio, P. Navas, I. Hernandez, P. Bedate, A. Moreno, A. Mendoza, A. Martinez, J. Palomino, F. Arribas and P. Escribano

Cardiology Departament, University 12 Octubre Hospital, Madrid, Spain; Medical and Molecular Genetic Institute University, La Paz Hospital, Madrid, Spain; CIBER Enfermedades cardiovasculares, Madrid, Spain; Cardiology Department, Gregorio Marañón Hospital, Madrid, Spain; Cardiology Department, Fundación Jimenez Diaz Hospital, Madrid, Spain; Respiratory Department, Asturias University Hospital, Asturias, Spain; Pediatric Department, Vall d’Hebron Hospital, Barcelona, Spain; Pediatric Department, University 12 Octubre Hospital, Madrid, Spain; Respiratory Department Universitary Valdecilla Hospital, Santander, Spain

Heritable forms of pulmonary arterial hypertension (PAH) and pulmonary veno-occlusive disease (PVOD) have different lung histopathological lesions. BMPR2 is the first and most frequent gene associated with PAH, with autosomal dominant trait. Histological features of PAH are medial hypertrophy of muscular and elastic arteries, dilation, and intimal atheromas of elastic pulmonary arteries, and in severe forms plexiform lesions. Homozygous mutation in EIF2AK4 is related to PVOD and associated with more pronounced venous and veins remodeling. The objective of this study was to describe clinical presentation and prognosis in a cohort of patients with pulmonary hypertension according to genetic etiology. We performed a prospective observational study in a cohort of patients with PAH included in the Spanish registry of PAH (REHAP), and we performed a genetic testing through next generation sequencing. We analyzed clinical characteristics of patients with BMPR2 (n = 23) and EIF2AK4 (n = 26) gene mutation. Results showed that patients with mutation in EIF2AK4 were younger at the diagnosis (24.9 vs. 32.7 years, p = 0.03) and did not have gender predilection. In addition, they had worse functional class (78% functional class III/IV vs. 43%, p = 0.02) and less distance covered in six minute walk test (349 vs. 370, p < 0.01). All carriers of EIF2AK4 had low diffusing capacity for carbon monoxide; however, the ones who carried BMPR2 did not show this disorder. BMPR2 carriers had more severe hemodynamic parameters (pulmonary vascular resistance 10.6 vs. 15.9 UW, p = 0.02). Despite that, survival free of transplant at three years was 42.8% in EIF2AK4 carriers vs. 95.2% in BMPR2 carriers (p < 0.01). In conclusion, patients who carried mutation in EIF2AK4 were younger, with worse functional class and poor survival, despite that, carriers of BMPR2 had more severe hemodynamic parameters.

Single-cell RNA sequencing reveals marked expansion of a discreet cluster of apelin-expressing endogenous lung endothelial progenitors after induction of widespread endothelial injury

Rafael S. Godoy, Nicholas Cober, Ketul Chaudhary, Yupu Deng, David Cook, Katelynn Rowe and Duncan J. Stewart

Sinclair Center for Regenerative Medicine, Ottawa Hospital Research Institute, Ottawa, Canada; Faculty of Medicine, University of Ottawa, Ottawa, Canada

Inhibition of VEGF receptors with SU5416 (SU) in rats results in widespread induction of endothelial cell (EC) apoptosis, which, when combined with transient exposure to hypoxia, produces a progressive model of severe pulmonary arterial hypertension (PAH). However, the mechanisms linking EC loss to occlusive arterial remodeling have not been delineated. We hypothesized that the balance between EC injury and repair is critical for the initiation and progression of PAH. We used single-cell RNA sequencing (scRNAseq; 10× Genomics) to identify changes in gene expression profiles in multiple cell populations within the lung before and one week after induction of EC injury by a single injection of SU (20 mg/kg). Using U-Map cluster analysis, we could identify 23 discrete clusters of cells in the uninjured lung based on transcriptional profiles, including 6 separate EC populations, the largest represented by microvascular (MV) ECs (73%), followed by venous (9%) and arterial ECs (4%) and a distant cluster with overrepresentation of genes related to proliferation and survival (5%), possibly indicative of an endothelial progenitor cell population. In addition, there was a secondary rare EC group (7%), with abundant expression of proliferation and survival genes, nested close to the MV population, also distinguished by high expression of Apelin, which plays an important role in angiogenesis. One week after SU alone, a marked shift EC gene expression was observed in the MV cluster, while the proportion of arterial ECs was uniquely reduced by 3.9-fold. Interestingly, the secondary progenitor cell cluster was the only population that expanded after SU by more than two-fold, suggesting a potential role in early lung microvascular repair. In conclusion, scRNAseq resolved six distinct lung EC populations including two novel clusters that likely represent pools of endothelial progenitors, one of which exhibited high Apelin expression and was uniquely expanded after discrete EC injury.

Relationship between pulmonary vascular pruning and perfusion defects in thromboembolic lung disease

Pietro Nardelli, Partha V. Hota, Eileen Harder, Gonzalo V. Sánchez-Ferrero, Aaron B. Waxman, George R. Washko, Andetta R. Hunsaker, Raúl S.J. Estépar and Farbod N. Rahaghi

Division of Pulmonary and Critical Care, Departments of Radiology, Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

The availability of dual-energy computed tomography (DECT) has allowed for more detailed study of lung perfusion. CT-based studies have demonstrated pruning in thromboembolic lung diseases as well as perfusion variability, but the relationship between pruning and perfusion variability is not well characterized. The purpose of this analysis was to study the variability in perfusion using DECT in a cohort of patients with and without thromboembolic disease and to compare this to pruning in the same subjects. All DECTs with complete original information that were performed on the Siemens platform were retrospectively identified. All DECTs (were reconstructed at high resolution (0.8 mm thickness slices) and the pulmonary vasculature was reconstructed from the same images, and the arterial and venous vasculature were labeled using a convolutional neural network, validated previously by manually labeling. Measures of pulmonary blood volume were then extracted, with exact sizing provided by a deep learning-based neural network. Venous and arterial BV5 and TBV (vessels < 5 mm² in cross-section and total pulmonary blood vessel volume) were measured. The BV5/TBV ratio, a marker of pruning, was calculated for the arterial and venous systems. There were 87 DECTs included in the final cohort including 19 subjects with chronic thromboembolic pulmonary hypertension (CTEPH), 11 subjects with chronic thromboembolic disease (CTED), 33 patients with acute pulmonary embolism (PE), and 24 subjects with no pulmonary thromboembolic disease by imaging (Fig. 1). Upon analysis of the entire cohort, increased aBV5/TBV correlated with a decreased standard deviation of the normalized iodine perfusion map voxel values (R = 0.54). A similar relationship was also seen in the venous BV5/TBV (R = 0.62). In summary, patients with more distal pruning tended to have greater variability in their perfusion imaging than those with mild pruning. This suggests that pulmonary vascular remodeling on a macroscopic scale is associated changes in lung perfusions in thromboembolic disease.

Nudix hydrolase 1 (NUDT1) promotes pulmonary arteries smooth muscle cells (PASMCs) adaptation to stressful environment and represents a promising target in pulmonary arterial hypertension

G. Vitry, O. Boucherat, M.C. Lampron, K. Habbout, Y. Grobbs, A. Bourgeois, R. Paradis, V. Nadeau, S. Breuils-Bonnet, E. Tremblay, S. Martineau, M. Orcholski, C. Lambert, S. Provencher, S. Bonnet and R. Paulin

Faculté de Médecine, Université Laval, Qebec, Canada; Axe Pneumologie, Centre de Recherche de l’IUCPQ, Qebec, Canada

Pulmonary arterial hypertension (PAH) is characterized by progressive pulmonary arteries (PAs) obstruction leading to increased blood flow resistance and heart failure. Pulmonary artery smooth muscle cells (PASMCs) of PAH patients display a “cancer-like” phenotype that contribute to PA remodeling. However, PAH-PASMCs very stressful environment should normally trigger excessive DNA damage accumulation and cell death. This suggests that PAH-PASMCs have developed adaptive mechanisms to face stress which might be interesting targets in PAH. NUDT1 is a DNA protective enzyme that hydrolyzes oxidized guanines (8OHdG), preventing DNA damage overload and cell death. Given the antitumoral effects of NUDT1 inhibition, we hypothesized that NUDT1 overexpression is essential for the cancer-like phenotype of PAH-PASMCs and vascular remodeling. We used primary cell lines and tissues from control and PAH patients as well as animal models of PAH and assess the effect of NUDT1 inhibition by siRNA and (S)-Crizotinib both in vitro and in vivo. We found by a proteomic study that NUDT1 is one of the most overexpressed proteins in PAH-PASMCs. Accordingly, NUDT1 expression measured by immunoblot was increased in PAH-tissues and PASMCs (p < 0.05) (n = 9), compared to control donors (n = 9), and also in animal models (monocrotaline (MCT), Sugen/Hypoxia, and Fawn Hooded rats, n > 5) (p < 0.05). NUDT1 inhibition in PAH-PASMCs resulted in (i) marked accumulation of 8OHdG in both nuclear and mitochondrial DNA (immunofluorescence, p < 0.01); (ii) excessive DNA damage (Comet Assays, p < 0.05); (iii) compromised bioenergetic functions (Seahorse, p < 0.05); (iv) impaired autophagic flux (Immunofluorescence/blot, p < 0.001), and finally (v) decreased proliferation/apoptosis resistance (Ki67/TUNEL, p < 0.01). In vivo, nebulization of (S)-Crizotinib to MCT rats with established PAH improved hemodynamics (right heart catheterization: RVSP, mPAP, CO, p < 0.05) and vascular remodeling (Elastica van Gieson staining, p < 0.05). In conclusion, NUDT1 overexpression in PAH-PASMCs represents a stress-buffering mechanism supporting cell survival and proliferation. Inhibition of NUDT1 may represent a novel therapeutic avenue for PAH.

Lymphatic thoracic duct diameter index magnetic resonance T2 weighted in pulmonary artery hypertension patients

Juaneda Ernesto, Catalfaro Danilo, Fregapani J. Pablo, Guevara Antonio, Peirone Alejandro, Juaneda Ignacio and Kreutzer Christian

Instituto Oulton, Hospital Privado and Hospital de Niños, Córdoba, Argentina

Lymphatic abnormalities have been described after functional single-ventricle palliation using magnetic resonance (MR) lymphangiography. Pulmonary artery hypertension (PAH) may occur in univentricular and biventricular circulation. PAH may increase systemic venous pressure impairing lymphatic drainage. The objective was to quantify thoracic duct diameter index (TDDI) in PAH patients. We retrospectively reviewed MR images (T2 weighted lymphangiography) of 11 patients with PAH, age 14.7 ± 8.6 years, between January 2017 and October 2019, performed with 3T Siemens MR, with respiratory navigator, without anesthesia/contrast enhancement. Patients were divided into two groups: four univentricular circulation (three unfenestrated Fontan–Kreutzer, one Glenn), seven biventricular circulation: five isolated PAH and two Eisenmenger síndrome (ES). PAH was defined on cardiac catheterization: univentricular circulation when transpulmonary gradient > 6 mmHg; biventricular circulation when mean pulmonary artery pressure > 20 mmHg, pulmonary vascular resistance > 3 Woods Units m². TDDI mm/m² was measured proximal to left venous yugulosubclavia junction. Real-time assessment MR cineangiogram ejection fraction was measured in all patients. Results showed that univentricular circulation included two patients with protein loosing enteropathy and one with plastic bronchitis; TDDI 4.8 ± 1.48 mm/m², transpulmonary gradient 7.75 ± 1.5 mmHg, and ejection fraction 53 ± 9%. Univentricular circulation TDDI when compared with isolated PAH and ES was significantly greater (p < 0.001). Biventricular circulation included three idiopathic, two hereditary, and two ES. TDDI: 2.32 ± 0.97 mm/m², mean pulmonary artery pressure: 56.5 ± 17.69 mmHg, pulmonary vascular resistance: 13.7 ± 7.2 WU/m², and ejection fraction: 55.8 ± 8.3%. Isolated PAH TDDI 2.54 ± 1.09 mm/m₂ vs. ES 1.78 ± 0.30 mm/m² (NS). In conclusion, lymphatic TDDI MR was significantly greater in univenticular circulation when compared with biventricular circulation PAH patients. Biventricular circulation PAH needs a greater number of patients and comparison with normal reference values in order to obtain statistical differences. Lymphatic thoracic duct MR T2 weighted may be useful in PAH cardiovascular MR protocol in order to quantify another risk stratification factor.

Description of lobar vascular volume and dimensions in a patient with scoliosis

Franck F. Rahaghi, Pietro Nardelli, George R. Washko, Raúl S.J. Estépar and Farbod N. Rahaghi

Department of Pulmonary and Critical Care, Cleveland Clinic Florida, Weston, FL, USA; Division of Pulmonary and Critical Care, Departments of Radiology, Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

Pulmonary hypertension has been described for over a century in the context of scoliosis. Possible mechanisms of pulmonary arterial hypertension in this cohort include hypoxemia due to restrictive lung disease but may also include an element of mechanical deformation of the pulmonary circulation. Here, we present a review of the history and data collected in patients with scoliosis. We discuss the case of a 60-year-old male with history of polio and scoliosis (Cobb angle 30°/Moderate Scoliosis) who is CPAP-dependent but remains dyspneic. Measures of pulmonary blood volume including BV5 (volume of vessels < 5 mm² in cross-section) and total pulmonary blood vessel volume (TBV) were then extracted from volumetric CT imaging using 3D vascular reconstruction. The distal volume (BV5) was normalized by TBV and lung volume (LV) for each lobe as a marker of pruning. BV5/TBV was 0.51 in the right lower lobe as compared to 0.41 in the left lower lobe. Similarly, the left upper lobe had a BV5/TBV of 0.48 in comparison. BV5/LV was 21.8 ml/L in the right lower lobe in comparison to 16.9 ml/L in the left lower lobe (Table 1). The loss of distal vasculature is shown in Fig. 1. Correlation between scoliosis and pulmonary hypertension has been described, including correlation between Cobb’s angle and RVSP in adolescent idiopathic Scoliosis, reported to be reversible upon surgical intervention. The exact pathophysiology of pulmonary hypertension in a patient with scoliosis is not clear. This report suggests that local changes in pulmonary vascular capacity and vascular dimension may play a role through compression and pruning of vasculature. In conclusion, measurement of pulmonary blood volume and vascular pruning may allow further understanding of mechanism of pulmonary vascular disease in scoliosis.

Table 1.

Blood volume/pruning measurement by Lobe.

	VB5_TBV	BV5_LV
Right upper lobe (RUL)	0.546119	24,932.07
Right middle lobe (RML)	0.489788	22,480.21
Right lower lobe (RLL)	0.506231	21,807.79
Left upper lobe (LUL)	0.476486	20,601.33
Left lower lobe (LLL)	0.411191	16,903.84
Right	0.517275	22,978.75
Left	0.447733	18,926.99
Whole	0.490724	21,384.12

Fig. 1.

Loss of distal vasculature in scholiosis.

Estrogen receptor alpha (ERα) protects against pulmonary hypertension (PH) development

Andrea Frump, Bakhtiyor Yakubov, James Hester, Annie Gensel, Amanda Fisher, Todd Cook, Jenny Cheng, Marcela Tabima, Naomi Chesler and Tim Lahm

Division of Pulmonary, Critical Care, Sleep and Occupational Medicine, Indiana University School of Medicine, Indianapolis, IN, USA; Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI, USA

Estrogen receptor alpha (ERα) is beneficial in the systemic vasculature, with loss-of-function mutations linked to the development of cardiovascular disease. However, the role of ERα in PH is incompletely studied. Loss of ERα exacerbates experimental PH. ERα was measured in pulmonary artery endothelial cells (PAECs) from PAH patients. ERα loss-of-function mutant Sprague-Dawley rats were generated via CRISPR-Cas9. In vivo studies were performed in male and female wild-type (WT) or ERα mutant rats with monocrotaline (MCT)- and SuHx-induced PH. Hemodynamics, PA remodeling, and molecular changes were assessed. PAECs and RV-specific endothelial cells (RVECs) were isolated from ERα mutant rats and evaluated by matrigel assay. To study effects of ERα activation, PPT (an ERα-selective agonist) was utilized in MCT-PH using a rescue approach. Results showed that PAH-PAECs patients tended to exhibit lower ERα expression compared to controls. In experimental PH, loss of ERα resulted in more severe disease (increased RV hypertrophy, RV systolic pressure (RVSP), total pulmonary resistance index, PA remodeling, and decreased cardiac index and TAPSE) compared to WT (p < 0.05). This difference in phenotypes was driven by more severe changes in females, whereas changes in male ERα mutants were less pronounced. Loss of ERα in isolated rat PAECs or RVECs significantly reduced ring formation (p < 0.05). Conversely, PPT rescued MCT-PH; ERα agonist treatment was associated with less-severe MCT-induced alterations in RVSP, RV hypertrophy, and cardiac structure/function (p < 0.05). ERα agonist treatment increased expression of homeostasis regulators BMPR2 and apelin in vitro and in vivo (p < 0.05). In conclusion, loss of ERα results in more severe PH. Selective ERα activation attenuates PH. These data suggest that ERα exerts vasculoprotective effects in PH. The more severe phenotype in female ERα mutants with PH suggests that alterations in ERα signaling may contribute to the female bias in PAH. Selectively activating ERα signaling may be a novel strategy to treat PAH.

Protein quantitative trait loci inform genetic risk of pulmonary arterial hypertension and novel candidate pathogenic pathways

Lars Harbaum^*, Christopher J. Rhodes^*, Allan Lawrie, Jason H. Karnes, Tae-Hwi L. Schwantes-An, Ankit A. Desai, William C. Nichols, Marc Humbert, David Montani, Barbara Girerd, H.A. Ghofrani, Friedrich Grimminger, Christian Gieger, Karsten Suhre, Johannes Graumann, Holger Schulz, Annette Peters, Mark Toshner, David G. Kiely, Luke S. Howard, Marika Kaakinen, Inga Prokopenko, Marta Bleda, Matthias Haimel, Charaka Hadinnapola, Emilia M. Swietlik, Stefan Gräf, Nicholas W. Morrell, John Wharton and Martin R. Wilkins

Faculty of Medicine, National Heart and Lung Institute, Imperial College London, London, UK; Department of Infection, Immunity and Cardiovascular Disease, University of Sheffield, Medical School, Sheffield, UK; Department of Pharmacy Practice and Science, University of Arizona College of Pharmacy, Tucson, AZ, USA; Department of Medical and Molecular Genetics, and Krannert Institute of Cardiology, Department of Medicine, Indiana University, Indianapolis, IN, USA; Division of Human Genetics, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH, USA; Faculté de Médecine, University Paris-Sud, Université Paris-Saclay, Le Kremlin Bicêtre, France; Department of Internal Medicine, Justus-Liebig-University Giessen, Giessen, Germany; Institute of Epidemiology, Helmholtz Zentrum München, Neuherberg, Germany; Department of Physiology and Biophysics, Weill Cornell Medicine-Qatar, Doha, Qatar; Department of Biomolecular Mass Spectrometry, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany; Department of Medicine, University of Cambridge School of Clinical Medicine, Cambridge, UK

^*These first authors contributed equally.

Idiopathic and heritable pulmonary arterial hypertension (PAH) are characterized by an imbalance of vasoactive factors, inflammation and disrupted cellular growth, leading to structural remodelling of pulmonary arteries and arterioles. Many of these processes are reflected in perturbation of circulating proteins. Recent advances in proteomic profiling and integration with genomic data have demonstrated that a large number of circulating proteins are under substantial genetic control. This insight has enabled strategies to test for causal interferences based on protein quantitative trait loci (pQTL). We compared the plasma proteome from patients with idiopathic, heritable PAH with that from control subjects using an aptamer-based assay. We established genome-wide significant pQTL through a meta-analysis of three genome-wide association studies (GWAS) including patients with PAH. Two complementary approaches were used to integrate pQTLs and genetic risk for PAH; genetic scores predicting protein levels were tested for association with PAH in two case-control cohorts and Mendelian randomisation studies were performed using an international GWAS of PAH. Results showed that of 1113 directly measured proteins, the concentrations of 111 were discovered and validated to be different between patients with idiopathic or heritable PAH and healthy volunteers. A subset of 77 proteins also distinguished PAH patients from a distinct population-based cohort. We detected pQTLs for 51 of these 77 proteins. The two integrated genomic approaches revealed seven candidate proteins contributing to risk for PAH. Genetically determined higher plasma levels of c-Met, VEGFR2, SOD2 and L1CAM were associated with decreased risk of PAH, while higher levels of DC-SIGN, ANGL4 and ASAH2 were associated with increased risk. Our findings provide new insights into the molecular characterisation of idiopathic and heritable PAH and establish a set of circulating proteins that can help to prioritise targets for experimental studies and drug development.

Response to vasodilator challenge across varying etiologies of pulmonary arterial hypertension

K. Kearney, N. Bart, A. Pideaux, K. Brown, C. Corrigan, E. Koppe, E. Kotlyar and A. Keogh

Heart and Lung Transplant Unit, St. Vincent’s Hospital, Sydney, Australia

Vasodilator provocative testing has traditionally been used to differentiate a group of idiopathic pulmonary arterial hypertension (PAH) “super responders” who have an excellent long-term prognosis and about half of whom respond to calcium channel blockers. Previous studies have shown few responders in other etiologies of PAH. Retrospective review of all nitric oxide vasodilator challenges performed at a single PAH center from 1999 to 2018. Statistics were performed in Excel. Results showed that there were 79 patients with nitric oxide challenges performed over the follow-up period. Five WHO Group 5 patients were excluded. There were 25 mortalities in this cohort. Table 1 shows the comparison of vasoreactivity across different etiologies, follow-up time, and mortality. Classic vasoreactivity was observed in both idiopathic and CTEPH patients. Despite not exhibiting a classic vasoreactivity response, substantial reductions in PVR were observed across all etiologies examined. In conclusion, classic vasoreactivity can be observed outside of idiopathic PAH patients. A substantial decrease in PVR at vasoreactive challenge can be observed across a variety of etiologies of PAH.

Table 1.

Vasodilator response and follow-up data.

Etiology	iPAH	HPAH	CTD	CTEPH	CHD	Total
n	34	4	12	17	7	74
Classic VR	5 (15%)	0	0	1 (6%)	0	6 (8%)
Decrease in PVR > 20%	21 (60%)	4 (100%)	8 (67%)	10 (59%)	3 (43%)	46 (62%)
Mean follow-up (years)	3.4	7.6	2.7	4.8	3.3	3.8
Mortality in classic responders	2 (40%)			0		2 (33%)
Mortality in non-responders	11 (32%)	1 (25%)	7 (88%)	6 (38%)	0	25 (34%)

iPAH, idiopathic pulmonary arterial hypertension; HPAH, hereditary pulmonary arterial hypertension; CTD, connective tissue disease; CTEPH, chronic thromboembolic pulmonary hypertension; CHD, congenital heart disease; PVR, pulmonary vascular resistance.

Defining vasoreactivity in idiopathic pulmonary arterial hypertension

K. Kearney, N. Bart, A. Pideaux, K. Brown, C. Corrigan, E. Koppe, E. Kotlyar and A. Keogh

Heart and Lung Transplant Unit, St. Vincent’s Hospital, Sydney, Australia

Vasodilator provocative testing has traditionally been used to differentiate a group of idiopathic pulmonary arterial hypertension (PAH) “super responders” who have an excellent long-term prognosis and about half of whom respond to calcium channel blockers. The optimal criteria for defining a responder and whether they should be treated with calcium channel blockers or modern PAH therapy is unclear. Markers of RV–PA coupling in context of vasodilator challenge remain unexplored. Retrospective review of all nitric oxide vasodilator challenges performed at a single PAH centre from 1999 to 2018 was performed. Statistics was performed in Excel. Classic vasoreactive response was defined as a mean MPAP > 10 mmHg to a MPAP < 40 mmHg without a decrease in CO. Pulmonary artery pulsatility index (PAPI), a measure of RV–PA coupling, was defined as systolic pulmonary artery pressure (SPAP) – diastolic pulmonary artery pressure (DPAP)/right atrial pressure (RAP). There were 79 patients with nitric oxide challenges performed over the follow-up period. Thirty-four of these were performed in patients with a diagnosis of idiopathic PAH. Five patients exhibited a classic vasoreactive response, 21 patients had a decrease in PVR by > 20% and 10 patients increased PAPI with vasodilator challenge. Kaplan–Meier survival curves for all three categories are shown (Figs. 1--3). In conclusion, there was no currently utilised or novel measure tested here that elicited a long-term mortality difference in idiopathic PAH undergoing vasodilator challenge.

Targeting AK4 for the regulation of the hypoxia-driven responses of pulmonary vascular cells implicated in the pathogenesis of pulmonary hypertension

Magdalena Wujak, Tessa Wilke, Cheng-Yu Wu, Ralph T. Schermuly, Hossein A. Ghofrani, Werner Seeger, Friedrich Grimminger and Norbert Weissmann

Cardio-Pulmonary Institute, Universities of Giessen and Marburg Lung Center, German Center for Lung Research, Justus Liebig University Giessen, Giessen, Germany; Faculty of Biological and Veterinary Sciences, Nicolaus Copernicus University in Torun, Torun, Poland

Endothelial dysfunction and vascular remodelling are major mechanisms contributing to the development and progression of pulmonary arterial hypertension (PAH). Pulmonary vascular cells in PAH exhibit a mitochondrial-metabolic phenotype similar to that seen in cancer, indicating that the knowledge gained from cancer research may help in the treatment of PH. Recently, adenylate kinase isoenzyme 4 (AK4) was demonstrated as a potent marker of poor clinical outcomes that promotes metabolic reprogramming and metastasis of lung cancer. However, the role of AK4 in the development of PH is completely unknown. The aim of our study was to investigate the impact of AK4 on hypoxia-driven responses of pulmonary artery smooth muscle cells (PASMC) and pulmonary microvascular endothelial cells (PMVEC) in order to evaluate this protein as a new possible target in PH. We found that AK4 is upregulated in both PASMC and PMVEC exposed to hypoxia in HIF-1α-dependent manner as well as in chronic hypoxia mouse model representing group III of PH classification. Furthermore, immunohistochemical staining of human lungs showed a significant increase in AK4 level in the pulmonary vasculature of IPAH patients as compared to donors. Using siRNA-mediated gene silencing technique, we found that AK4 downregulation in PASMC slightly increases cell proliferation but markedly improves the mitochondrial respiratory function reflected as the oxygen consumption rate measured with Seahorse approach, whereas AK4 silencing in PMVEC leads to decrease in apoptosis through the inhibition of ERK1/2 phosphorylation and caspase 3/7 activity. Our findings indicate that AK4 might play a distinct role in different types of pulmonary cells, and AK4 targeting might be a novel approach to modulate hypoxia-driven responses in the pulmonary vasculature. (This study was supported by the European Respiratory Society, and the Deutsches Zentrums für Lungenforschung, Fellowship LTRF 2018).

Prevalence and incidence of pulmonary arterial hypertension: a systematic review of the literature for the Global Burden of Disease study

Sophia Emmons-Bell, Alie B. Dooley, Catherine Johnson and Gregory Roth

Division of Cardiology, Institute for Health Metrics and Evaluation, University of Washington, Seattle, WA, USA

The Global Burden of Disease (GBD) study is a multinational effort to produce comparable, consistent measures of disease burden for populations, but pulmonary arterial hypertension (PAH) is not included. An increasing number of studies describe the epidemiology of PAH. We sought all data on population-based prevalence, incidence, and mortality to estimate the global burden of PAH for the GBD study. We searched the Global Index Medicus, including PubMed and other databases, for key words between 1980 and 2020, identifying population-representative prevalence, incidence, and mortality rates of clinically diagnosed PAH supported by diagnostic testing. We excluded PAH groups 2–5, referral center cohorts, or study samples < 50. The search returned 10,098 hits, of which 56 were included: 13 for prevalence, 16 for incidence, and 50 for case fatality rate (23 contained > 1 measure). The mean age of patients was 47 years, with 67.5% female; 27 countries from six GBD super-regions were represented. Reported prevalence ranged from 0.37 cases/100,000, derived from a referral center of French children, to 4.9 cases/100,000, derived from a Swedish register. Reported incidence ranged from 0.07 cases/100,000 person-years, derived from a study in the UK and Ireland, to 0.56 cases/100,000 person-years, derived from an Israeli clinic. All studies with sex-specific estimates reported higher levels in females than males. Studies varied in their approach to reporting age–sex categories, and in sampling procedures. Reported PAH prevalence, incidence, and mortality rates varied widely by location and study. Harmonization of methods for PAH registries would improve efforts at disease surveillance. Results of this search can be used to inform planned GBD estimation of PAH and help to quantifying its global burden, thereby guiding global efforts to prioritize and treat this often-overlooked condition.

Global death rates of pulmonary arterial hypertension: an analysis for the Global Burden of Disease study

Sophia Emmons-Bell, Catherine Johnson and Gregory Roth

Division of Cardiology, Institute for Health Metrics and Evaluation, University of Washington, Seattle, WA, USA

The Global Burden of Disease (GBD) 2017 study provides estimates of cause-specific mortality for 282 causes from 1980 to 2017, but does not include pulmonary arterial hypertension (PAH). As part of the GBD 2020 study, we used all available cause-specific death data to estimate the global and regional patterns of PAH mortality. The GBD mortality database includes vital registration, verbal autopsy, registry, and surveillance data. For each data source, underlying causes of death were mapped from International Classification of Disease (ICD) codes to GBD causes. ICD codes 416.0, I27.0, and I27.2 were mapped to PAH. Deaths coded to nonspecific or inappropriate causes such as senility were redistributed across all causes, including a small fraction to PAH. We used the Cause of Death Ensemble model to estimate death rates across all location-years. Results showed that between 1990 and 2019, there were 4.54 (95% uncertainty interval 3.15–5.80) million deaths due to PAH, of which 2.62 (1.58–3.63) million were female. The all-age death rate in 2019 was 2.52 (1.61–3.04) per million males and 3.81 (1.99–4.91) per million females, an increase of 74% and 150%, respectively, from 1990 rates. Across all years, the death rate among females was 34% higher than among males. The highest estimated rates in 2019 were in the United States and Bermuda. Healthcare access and quality index, sociodemographic index, and HIV prevalence were predictive for estimating death rates due to PAH. In conclusion, deaths due to PAH have increased since 1990, with the majority concentrated in women and high-income countries. Comparable estimates of PAH mortality across time and geography provide insight into the epidemiology of this rare disease.

Hemin induced endothelial dysfunction and endothelial to mesenchymal transition contributes to the pathogenesis of pulmonary hypertension due to chronic hemolysis

Dustin R. Fraidenburg

Division of Pulmonary, Critical Care, Sleep, and Allergy, Department of Medicine, University of Illinois at Chicago, Chicago, IL, USA

Pulmonary hypertension (PH) is a severe complication and independent predictor of mortality in sickle cell disease (SCD). Hemolysis and nitric oxide scavenging contribute to endothelial dysfunction and vascular remodeling leading to PH in SCD, yet the precise pathogenesis remains incompletely understood. We hypothesize that products of intravascular hemolysis lead to inflammation, increased endothelial proliferation, endothelial to mesenchymal transition (EndoMT), and ultimately contribute to the development and progression of PH in SCD. All experiments in the current study utilized human pulmonary artery endothelial cells (HPAEC). Cell proliferation was determined by WST assay (Millipore) and proliferating cell nuclear antigen (PCNA) expression. Migration was determined by wound assay using electric cell-substrate impedance sensing. Interleukin (IL)-6 and IL-8 secretion were evaluated by ELISA assay (BioLegend). Western blot analysis was used to quantify protein expression. Statistical analysis utilized t-test and ANOVA where appropriate. Results showed that HPAEC treated with 5 µM hemin showed increased proliferation (p < 0.001) and migration (p < 0.05) compared to control. HPAEC treated with hemin also showed increased IL-6 (p < 0.001) and IL-8 (p < 0.001) secretion. Increased protein expression of mesenchymal tissue markers, vimentin and alpha smooth muscle actin, were identified in HPAEC treated with hemin compared to control. Given the suggestion of increased EndoMT in HPAEC treated with hemin, we undertook experiments using an MLCK inhibitor, ML-7. Pretreatment with ML-7 almost fully prevented the increased proliferation seen in hemin-treated HPAEC (p < 0.01) and inhibited secretion of IL-6 and IL-8 (p < 0.001 for both). In conclusion, low dose (5 µM) hemin exposure leads to dysfunction of HPAEC characterized by increased proliferation and migration as well as cytokine release. Hemin treatment was associated with EndoMT as a potential mechanism for the development of PH in patients with chronic hemolysis. Targeting myosin light-chain kinase (MLCK) activity in HPAEC undergoing EndoMT may be a unique target in the early stages of PH development.

Vascular pruning and dual energy parenchymal voxel values as biomarkers of pulmonary thromboembolic disease

Farbod N. Rahaghi, Partha V. Hota, Pietro Nardelli, Eileen Harder, Gonzalo V. Sánchez-Ferrero, Andetta R. Hunsaker, Aaron B. Waxman, George R. Washko and Raúl S.J. Estépar

Department of Radiology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA; Division of Pulmonary and Critical Care Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

Dual energy CT imaging allows the estimation of relative parenchymal iodine concentration in different parts of the lung—a marker that is used to detect perfusion defects in pulmonary thromboembolic disease. Vascular pruning has also been proposed as a marker of pulmonary vascular disease in thromboembolic disease. In this study, we examine the trends in measurements from the iodine maps and vascular reconstructions from a cohort of patients with dual energy CT imaging. DECTs with complete original information performed on the Siemens platform were retrospectively identified. Voxel values were extracted from the iodine maps sampled at 0.8 mm thickness, inside of the lung, with the vasculature removed. The pulmonary vasculature was reconstructed, and the arterial and venous vasculature was labeled using a convolutional neural network. Measures of pulmonary blood volume were then extracted, including BV5 (vessels < 5 mm² in cross-section), BV10, and total pulmonary blood vessel volume (TBV) for both the pulmonary arterial and venous systems. The BV5/TBV and BV10/TBV ratio, markers of pruning, were extracted from the CT. Results showed that 29 patients without thromboembolic disease, 18 patients with chronic thromboembolic pulmonary hypertension (CTEPH), and 13 patients with chronic thromboembolic disease (CTED) were identified for total of 60 subjects. There was a trend toward decreased mean normalized voxel value between the patients with CTEPH and controls, but no difference in the standard deviation of voxel values (Fig. 1). There was increasing arterial vascular pruning going from controls (aBV10/TBV 0.65), CTED (aBV10/TBV 0.63), and CTEPH (aBV10/TBV 0.55). Within the CTEPH cohort where pulmonary vascular resistance (PVR) values were available, the median and standard deviation of voxel values and venous BV10/TBV correlated with PVR. In conclusion, vascular volume and voxel iodine-based statistics derived from dual energy imaging may provide complimentary information about pulmonary vascular remodeling and perfusion abnormalities in pulmonary thromboembolic disease and can both be derived from dual energy CT imaging.

Impaired efficiency of right ventricular energetics in maladaptive right ventricular remodeling in response to pressure overload in Fischer rats

Jason G.E. Zelt, Virgilio J. Cadete, Yupu Deng, Mohammad Abdul-Ghani, Lynn A. Megeney, Robert deKemp, Rob Beanlands, Duncan J. Stewart and Lisa M. Mielniczuk

Division of Cardiology, Department of Cellular and Molecular Medicine, Heart Institute, Molecular Function and Imaging Program, The National Cardiac PET Centre, University of Ottawa, Ottawa, Canada; Sinclair Centre for Regenerative Medicine, Ottawa Hospital Research Institute, Ottawa, Canada; SPrott Centre for Stem Cell Research, Ottawa Hospital Research Institute, Ottawa, Canada

There is heterogeneity in right ventricular (RV) remodeling and the likelihood of progression to RV failure (RVF) in response to pulmonary arterial hypertension (PAH) that is not explained by differences in the degree of afterload. We aimed to characterize alterations in RV metabolism during the development of RVF secondary to PAH in Sprague-Dawley (SD) and Fischer rats that exhibit adaptive and maladaptive RV remodeling, respectively. PAH was induced by a subcutaneous injection of the VEGFR antagonist, SU5416, and three-weeks of hypoxia (SuHx). Fischer rats exhibited 100% mortality by five-weeks, whereas SD rats showed excellent survival up to nine weeks (88%; p < 0.0001). Proteomics analysis revealed 92 differentially expressed RV proteins in Fischer compared to SD PAH rats (p < 0.05); of these, 27% were mitochondrial, suggesting a role for mitochondrial energy metabolism as an early determinant for maladaptive RV hypertrophy. At four weeks, in vivo oxidative metabolism, assessed by [¹¹C] acetate clearance (kmono), was significantly elevated in Fischer rats compared to SD (RV/LV ratio: 0.82 ± 0.09 vs. 1.0 ± 0.11 min⁻¹, respectively; p < 0.05). Despite higher oxidative metabolism, Fischer rats exhibited a trend toward reduced RV power (p = 0.055), and therefore, impaired RV efficiency compared to SD (0.24 ± 0.04 vs. 0.45 ± 0.11J/s × min⁻¹ × g, respectively; p < 0.001). The potential for elevated oxidative metabolism in Fischer was also confirmed ex vivo by citrate synthase activity in RV homogenates. Activity of this key tricarboxylic acid enzyme was markedly elevated in both naïve and SuHx Fischer in comparison to SD rats. In contrast, no baseline differences were observed in oxidative phosphorylation complex activity, measured in permeabilized RV cardiac fibers. Together, these data suggest inefficiency in Fischer rats is due to impaired coupling between oxidative metabolism and ATP production. In conclusion, these data point to impaired efficiency of RV energetics in the development of maladaptive remodeling in Fischer rats, and suggest that strategies to improve RV energetics may reverse this maladaptive phenotype.

The mitochondrially located proteins, p66shc and cyclophilin D, are contributing to the regulation of the pulmonary vascular tone

Mareike Gierhardt, Julia Schäffer, Oleg Pak, Akylbek Sydykov, Karin Quanz, Azadeh Esfandiary, Jochen Wilhelm, Hossein A. Ghofrani, Ralph T. Schermuly, Werner Seeger, Friedrich Grimminger, Christiane Herden, Rainer Schulz, Norbert Weissmann and Natascha Sommer

Excellence Cluster Cardiopulmonary System, University of Giessen and Marburg Lung Center, German Center for Lung Research, Institute of Veterinary Pathology, Institute of Physiology, Justus Liebig University, Giessen, Germany

Mitochondria regulate intracellular calcium homeostasis and thereby may affect pulmonary vascular functions, in particular the response of the pulmonary vasculature to hypoxia. We investigated whether the proteins, p66shc and cyclophilin D (CypD), which regulate release of mitochondrial reactive oxygen species and apoptosis, may influence pulmonary vascular functions. Mice deficient for p66shc (p66shc^–/–), CypD (CypD^–/–), or both proteins (p66shc/CypD^–/–) exhibited a lower pulmonary vascular resistance compared to wild-type (WT) mice determined in vivo by hemodynamic measurements and in isolated lungs. Systemic arterial pressure was, however, only lower in CypD^–/– mice. Pulmonary vasoconstriction and intracellular calcium increase induced by KCl was lower in the lungs or pulmonary arterial smooth muscle cells from gene-deficient compared to WT mice. However, cardiac function and pulmonary vascular morphometric parameters did not differ. All mouse strains showed a similar response to chronic hypoxia with regard to development of pulmonary hypertension. We conclude that p66shc specifically regulates the pulmonary vascular tone, while CypD also affects systemic blood pressure. The pulmonary effects can be explained by decreased pulmonary vasoconstriction, suggested to be caused by p66shc and CypD interfering with the intracellular calcium homeostasis. As the observed differences were not additive in the p66shc/CypD^–/– double ko mice, p66shc and CypD most likely act via similar pathways. In conclusion, p66shc thus may be a new candidate for targeting pulmonary vascular disease. (Supported by Deutsche Forschungsgemeinschaft Grant SFB1213, Project A06 and B05).

Evidence of responders and non-responders to specific drugs for treatment of pulmonary arterial hypertension in Colombia: the experience of the “Colombian Network of Pulmonary Hypertension”

Mauricio Orozco-Levi, Melissa Mogollón, Javier Fajardo, Alba Ramírez-Sarmiento, Rafael Conde, Héctor Ortega and Manuel Pacheco

Alejandro Londoño; and the HAPred.co.

Servicio de Neumología, Centro para el Cuidado de la Salud Respiratoria, Hospital Internacional de Colombia, Universidad de Santander, Santander, Colombia; Universidad Industrial de Santander, Bucaramanga, Colombia; IPS Respiremos, Pereira, Colombia; Clínica CardioVid, Medellín, Colombia

Fundación Neumológica Colombiana, Bogotá, Colombia

Pulmonary arterial hypertension (PAH) is a chronic multifactorial hemodynamic disorder that imposes a serious impact on the patient and his environment as well as the national health system. In Colombia, specific drug therapy (oral, inhaled, or parenteral) offers 14 specific PAH drugs which represent 84 possible drug combinations for three therapeutic pathways (i.e. endothelin, nitric oxide, and prostacyclin). There is currently a multidisciplinary and multicenter initiative that consolidates the information of patients with hemodynamically confirmed diagnosis of PH (with emphasis on Groups 1 and 4) called the Colombian Network of Pulmonary Hypertension (HAPred.co) which allowed the analysis of the response to specific treatments in 503 patients in our country. The objective of this study was to evaluate the potential changes of pulmonary artery pressure and other hemodynamic or functional changes related to chronic treatment with specific drugs for PAH in incident patients of the disease (groups 1 and 4) in Colombia. The onset of treatment and its duration were analyzed as independent variables, and while pulmonary artery pressures (systolic, diastolic, and mean PAP) were included as dependent variables. Models of linear and non-linear correlation were analyzed regarding the change both in terms of absolute values ​and in percentage of change (post-pre/pre,%) between the invasive hemodynamic study (diagnosis-referent) and the last available catheterization in the tracking (final). The analysis was performed in 503 adult patients (50 ± 17 years, 74% women), with diagnosis of PH group 1 or 4. The relative change with treatment showed a wide range: PAPs decreased –9 ± 26%, PAPd = –18 ± 42%, and PAPm = –5 ± 31%. This dispersion was justified by the existence of three groups of patients: some without change in pulmonary pressures (i.e. non-responders), others with worsening of pressure (also non-responders), and others (50% of the total) showing an improvement (decrease of 10% or more of the initial value) of the mPAP (Figs 1 to 3). A linear function of correlation with the treatment time was not evident (p = ns, r² ≤0.1). Adjustment according to age showed no correlation with improvements. In conclusion, in patients with PAH in the Colombian Andean region, the response to specific approved drugs is not homogeneous in magnitude or sense in all patients. Responder patients (approximately 50%) and other non-responders are identified in terms of pulmonary artery pressure regardless of the time receiving treatments. This evidence highlights the need to objectively (invasively) evaluate pulmonary hemodynamics to identify patients who will require staging strategies or treatment combinations, including lung transplantation. (This study was supported, in part, by Grant BAYER HAPred.co 2019).

Fig. 1.

Kaplan Meier survival curve comparing those with a decrease in PVR > 20% (VR) to those who did not (non-VR).

Fig. 2.

Kaplan Meier survival curve for those exhibiting a classic vasodilator response (VR) to those who did not (non-VR).

Fig. 3.

Kaplan Meier survival curve for those increasing PAPI at vasodilator challenge compared to those who did not.

Dyspnea in patients with pulmonary hypertension is not a good predictor of disease severity: the experience of the “Colombian Network of Pulmonary Hypertension”

Mauricio Orozco-Levi, Melissa Mogollón, Javier Fajardo, Alba Ramírez-Sarmiento, Rafael Conde, Héctor Ortega and Manuel Pacheco

Alejandro Londoño; and the HAPred.co.

Servicio de Neumología, Centro para el Cuidado de la Salud Respiratoria, Hospital Internacional de Colombia, Santander, Colombia; Universidad de Santander, Bucaramanga, Colombia; Universidad Industrial de Santander (UIS), Bucaramanga, Colombia; IPS Respiremos, Pereira, Colombia; Clínica CardioVid, Medellín, Colombia; Fundación Neumológica Colombiana, Bogotá, Colombia

Pulmonary hypertension (PH) is a severe hemodynamic disorder in which multiple variables have been associated with severity and prognosis of patients. In the clinical arena, symptoms of dyspnea, functional class, and exercise capacity (walking or cycloergometry test variables) are commonly used to classify patients in low-, medium-, or high-risk mortality categories. Nevertheless, our clinical perception within the Colombian Network of Pulmonary Hypertension (HAPred.co) suggests that symptoms reported by patients of the Andean region could not linearly reflect the severity of the disease. The objective of this study was to assess the potential association between objective functional variables (hemodynamic and exercise date) with dyspnea or peripheral (legs) symptoms in patients with an incident diagnosis of groups 1 and 4 HP and living in the Andean region of Colombia. The information of all the patients (n = 503) identified within the Colombian Network of Pulmonary Hypertension (HAPred.co) was included in the analysis. The network is a multidisciplinary and multicenter trade union initiative that consolidates and analyzes the information of patients with hemodynamically confirmed diagnosis of PH, with special emphasis on Groups 1 and 4 of the disease. Correlation analyses (linear and non-linear) were performed between functional variables at rest (pressure, resistance, and cardiac output in right catheterization) and symptoms in various clinical evaluations. To date, we have included in the analysis 503 adult patients (50 ± 17 years, 74% women), with diagnosis of PH groups 1 and 4. The elapsed time between beginning of symptoms and diagnosis was 29 months (median 12 months, 10 patients with more than five years). A total of 67 patients had the diagnosis of PH group 1, the other group 4. In 78% of patients, one or more comorbidities exist. The functional class (NYHA) showed no linear correlation with age, BMI, echocardiographic, or pulmonary catheterization variables recorded at the time of diagnosis or the values at the last follow-up visit (p = ns, r² ≤0.1). In patients living in the Colombian Andean region, the functional class does not have a linear correlation with the severity of the hemodynamic variables during pulmonary catheterization. These results highlight the importance of objective evaluations in patients with PH of groups 1 and 4, since subjective variables (symptoms) at diagnosis or follow-up can drastically underestimate the degree of cardiac functional impairment. (This study was supported in part by Unrestricted BAYER grant HAPred.co, 2019).

Pressures underestimate the severity of cardiac output impairment in patients with pulmonary hypertension at the time of the diagnosis: the experience of the Colombian Network of Pulmonary Hypertension

Mauricio Orozco-Levi, Melissa Mogollón, Javier Fajardo, Alba Ramírez-Sarmiento, Rafael Conde, Héctor Ortega, Manuel Pacheco, Luis Echeverría, Jaime Rodríguez, Federico Saaibi and Alejandro Londoño

Pneumology Department, Center for Respiratory Health Care, Hospital Internacional de Colombia, Santander, Colombia. Universidad de Santander, Bucaramanga, Colombia; Universidad Industrial de Santander (UIS), Bucaramanga, Colombia; IPS Respiremos, Pereira, Colombia. Clínica CardioVid, Medellín, Colombia; Fundación Neumológica Colombiana, Bogotá, Colombia

The severity (morbidity and mortality) implicit in pulmonary hypertension (PH) requires close monitoring of patients to characterize their evolution and define specific therapy. We have recently described the absence of correlation between symptoms and hemodynamic data at the time of the diagnosis in patients with PH. Since the definition and monitoring of PH implies the recording of pulmonary pressures, we wanted to identify the potential correlation that these may have with the severity of the hemodynamic disorder in terms of cardiac output in patients residing in the Andean region. The objective of this study was to evaluate the association that may exist at the time of diagnosis between invasive hemodynamic data (right catheterism) and integral functional data (exercise capacity and cardiac output) in patients residing in the Andean region with an incident diagnosis of HP groups 1 and 4. The information of all the patients registered to date in the Colombian Network of Pulmonary Hypertension (HAPred.co) was included in the analysis. This multidisciplinary and multicenter gremial initiative consolidates and analyzes the information of patients with hemodynamically confirmed diagnosis of PH, with special emphasis on groups 1 and 4 of the disease. Correlation analyzes (linear and non-linear) of main resting hemodynamic variables (pressures, resistances, vasoreactivity test) were performed with six walking test, peak consumption of O₂ in cycloergometry, or cardiac output recorded in right catheterism. The analysis was performed in 104 adult patients (50 ± 17 years, 74% women), with diagnosis of HP groups 1 and 4. The variables of systolic, diastolic, and mean pressure did not show a mathematical function of correlation with functional variables (tolerated load, peak oxygen consumption, TM6) or cardiac output or cardiac index measured invasively (p = ns, r² ≤0.1); 100% of the cases showed a low heart rate. The age adjustment showed no improvement in the association. The reported time of previous symptoms until diagnosis was 29 months (median, 12 months, 10 patients with more than 5 years). A total of 67 patients presented a diagnosis of PH group 1, the other group 4; 78% of the patients had one or more comorbidities. In patients of the Colombian Andean region with PH, hemodynamic conditions are bad at the time of the diagnosis (cardiac index low in 100% of the patients). Pulmonary pressures do not correlate with the severity of changes in pulmonary catheterization or functional capacity before exercise. This evidence highlights the need to objectively (invasively) evaluate cardiac output in patients with PH of groups 1 and 4 in both diagnosis and follow-up to accurately assign specific therapies and avoid inappropriate prescription due to excess or defect of the same. (Supported, in part, by unrestricted Grant BAYER, 2019).

Treatment with oral treprostinil improves hemodynamics in participants with PAH

R.J. White, G. Meyer, T. Pulido, S.D.C. Jerjes, A. Khan, C.Q. Deng, R. Grover, M. Broderick, A. Ousmanou, L. Holdstock and E. Michelakis

University of Rochester Medical Center, Rochester, NY, USA; University of Alberta, Edmonton, Canada

Oral treprostinil (TRE) has been shown to improve exercise capacity and delay disease progression in patients with pulmonary arterial hypertension (PAH). Its effect on hemodynamics is unknown. FREEDOM-EV was an event-driven, Phase 3b, randomized, double-blind, placebo-controlled study. Dosing of study drug was titrated from 0.125 mg three times daily (TID) up to a maximum dose of 12 mg TID. All participants were using one approved PAH therapy at study entry, and some (n = 61) consented for a hemodynamics sub-study with right-heart catheterization at Baseline and Week 24. Participants with missing or mismatched cardiac output estimates (using indirect Fick or Thermodilution methodology) at Baseline and Week 24 were excluded from the analysis (n = 7). Analysis of covariance was used to measure differences with change from baseline in log-transformed data in hemodynamic parameter as the dependent variable, treatment as fixed effect, and log-transformed baseline hemodynamic parameter as a covariate. Results showed that there was a 19% reduction in mean pulmonary vascular resistance (PVR) from Baseline to Week 24 in the TRE group compared to a 1% reduction in the placebo group (–134.09 vs. –9.20; p = 0.0241). There was a corresponding 8% increase in cardiac output from Baseline to Week 24 in the TRE group compared to a 6% reduction in the placebo group (0.42 vs. –0.30; p = 0.0051). There were no significant changes in either mean right atrial pressure, mean pulmonary artery wedge pressure, or mean pulmonary artery pressure (PAPm) between the TRE and placebo groups. In conclusion, there was a significant reduction in PVR with an associated increase in cardiac output in the TRE group compared with the placebo group. Because there was no change in the PAPm, these data could be explained by the known pulmonary vasodilator effect of prostacyclin therapies, with or without a direct inotropic effect.

Parameters	Oral treprostinil				Placebo				p-Values
	n	Mean (Baseline)	Mean (Week 24)	Change from Baseline	n	Mean (Baseline)	Mean (Week 24)	Change from Baseline
RAPm (mmHg)	34	7.68	8.00	0.32	27	7.89	6.96	–0.93	0.8173
PAPm (mmHg)	34	49.15	46.03	–3.12	27	49.37	46.96	–2.41	0.5746
PAWPm (mmHg)	34	9.97	10.71	0.74	26	9.23	8.62	–0.62	0.1154
CO (L/min)	30	5.30	5.72	0.42	24	4.82	4.52	–0.30	0.0051
PVR (dynes*s/cm5)	30	720.23	586.14	–134.09	24	781.41	772.2	–9.20	0.0241

RAPm, mean right atrial pressure; PAPm, mean pulmonary artery pressure; PAWPm, mean pulmonary artery wedge pressure; PVR, pulmonary vascular resistance.

Medical cost of the drug treatment for pulmonary arterial hypertension in colombia

Mauricio Orozco-Levi, Alejandro Londoño, Rafael Conde, Ludy A. Parada, Ruben M. Figueroa and Camilo A. Leon

HAPRed.Co

Hospital Internacional de Colombia, Floridablanca, Columbia; CardioVID, Medellín, Columbia; Fundación Neumológica Colombiana, Bogotá, Columbia; BAYER, Leverkusen, Germany

In Colombia, no record exists regarding the potential cost of treatment for patients with pulmonary arterial hypertension (PAH) receiving single, dual or triple combination of specific drugs. It is necessary to evaluate these medical care costs due that new modules have appeared in the line of the recent Nice Guidelines for PAH. The objective of this study was to estimate the ceiling cost of medical treatment of patients with PAH in Colombia, according to the pharmacological possibilities existing in the country in line with the 2018-Nice Guidelines. The cost of the specific pharmacological treatment was estimated by modelling a tracer for adult patients (estimating 60 kg weight) submitted to drug therapy due to diagnosis of PAH (group 1 or 4). Ceiling estimations consider 100% of access, 100% adherence and 100% of tolerance to several pharmacological options available in Colombia in 2019. The information frequency of use and cost units of pharmacological treatment was obtained from a National Drug Price Information System (SISMED, official source of prices of medicines in Colombia) for IIIQ of 2018. Maximum, minimum and mean price ranges were defined in order to include market behaviour. The cost was expressed in both $COP and $USD. The difference between maximum and minimum represented the range. Normalization of the cost as proportion of Current Minimum Legal Salary (CLMS) was also performed in order to allow both future and international comparisons. Results showed that the current drug therapy of PAH in Colombia can include four pharmaceutical groups defined according to the action mechanisms: ERA (Bosentan, Ambrisentan, Macitentan), PDE5i (Sildenafil, Tadalafil) or GCs (Riociguat), and prostanoids (Iloprost, Treprostinil, Epoprostenol). The model was developed in line with both the recommendations of the international guidelines for the management of PAH and the common current clinical practice to estimate the cost of treatment using monotherapy, double combinations or triple combinations. Drug monotherapy discloses a mean annual cost of $66,800 USD (equivalent to 265 CLMS); a double combination $125,765 USD (501 SMLV); and a triple combination $200,130 USD (798 SMLV). The cost of medical treatment is inversely correlated with the percentage of adherence, tolerance and access to the medications, together with the actual percentage of patients under treatment. The cost of PAH drug treatment in Colombia imposes an extremely high cost to the national health system. This impact will increase significantly due to the improvements in the detection, treatment, adherence and survival of patients with PAH, as it will increase the number of patients and the duration of these specific treatments. This prognosis makes the indication, prescription and administration of these medications a priority for improvement through integrated medical actions such as HAPred.com and the national Colombian health system responsibilities. (This study was supported in part by unrestricted BAYER grant, HAPred.co 2019).

Restoration of lung microcirculation after unilateral pulmonary artery banding in a rat model of severe pulmonary arterial hypertension reveals a remarkable capacity for lung microvascular repair and regeneration

Yupu Deng^*, Ketul Chaudhary^*, Rafael S. Godoy, Anli Yang, Katelynn Rowe and Duncan J. Stewart

Sinclair Center for Regenerative Medicine, Ottawa Hospital Research Institute, Ottawa, Canada; Faculty of Medicine, University of Ottawa, Ottawa, Canada

^*These first authors contributed equally.

Pulmonary arterial hypertension (PAH) is triggered by endothelial cell (EC) apoptosis leading directly to loss of microvasculature and indirectly to occlusive arterial remodeling, together resulting in a profound loss of functional microcirculation. We hypothesized that the hemodynamic abnormalities of PAH, in particular increased luminal shear stress, play a vital role in perpetuating EC injury and ongoing EC apoptosis, overwhelming endogenous mechanisms of microvascular repair. Severe PAH was induced by the VEGF inhibitor, SU5416 (SU, 20 mg/kg), together with a three-week exposure to hypoxia (10% O², SuHx model) in SD rats. The left pulmonary vascular bed was protected from the effects of elevated arterial pressures and endothelial shear stress by left pulmonary arterial banding (LPAB > 70% stenosis), either at one-week prior to induction of PAH (prevention) or five-weeks after SU (reversal). Using micro-CT, we demonstrated > 80% loss in functional microcirculation by four weeks post SU, preceding evidence of occlusive arterial remodeling, and this was associated with persistent EC apoptosis up to seven weeks. Ongoing EC apoptosis and complex arterial remodeling at seven-weeks were abrogated by LPAB in the prevention model, with complete preservation of the lung microcirculation. Similarly, LPAB at five weeks resulted in resolution of EC apoptosis and arterial remodeling, which was associated with progressive recovery of the microcirculation at 8 and 10 weeks. Moreover, removal of the band at 10 weeks resulted in a 26 mmHg decrease in right ventricular systolic pressure three weeks later (81 ± 10 vs. 107 ± 10 mmHg; p < 0.05). In conclusion, two distinct mechanism are required for the development and progression of severe PAH in the SuHx model: the initial EC injury by SU and ongoing EC injury and apoptosis dependent on increased endothelial shear forces; the interruption of which by LPAB reveals a remarkable ability for lung microvascular repair and leads to reversal of established PAH.

Low potential for metabolic drug–drug interactions (DDIs) between rodatristat ethyl and approved medications for pulmonary arterial hypertension

Michelle Palacios, Katelyn R. Crizer, Eric Gaukel and Stephen Wring

Department of Translational Medicine, Altavant Sciences, Inc., Cary, NC, USA; Nonclinical Development, Roivant Sciences, Inc., New York, NY, USA

Rodatristat ethyl is an oral prodrug for rodatristat, a tryptophan hydroxylase inhibitor (lowers peripheral serotonin) in clinical development for pulmonary arterial hypertension (PAH). Combination therapy with rodatristat ethyl and ambrisentan or tadalafil in PAH rats yielded synergistic effects lowering mPAP and reducing vascular remodeling. Predicted maximum plasma concentrations (Cmax) in ELEVATE1 were compared to in vitro CYP450 interaction data to assess potential for systemic DDIs with PAH medications using information in product monographs. Rodatristat ethyl did not meaningfully inhibit (IC50) CYPs 2B6 (13 µM), 2C8 (2.8 µM), or CYP3A4/5 (≥13 µM). CYPs 1A2, 2C9, 2C19, or 2D6 were unaffected (IC50 > 30 µM). Predicted clinical Cmax for rodatristat ethyl (600 mg BID) is 0.25 µM, ∼10-fold below the IC50 of the most sensitive CYP (2C8) indicating low risk. Rodatristat inhibited 2C8 (2.8 µM, predicted plasma Cmax 2.01 µM). The ratio (0.014) of unbound Cmax (0.02 µM) to Ki (1.4 µM) indicates low potential for a clinically-relevant interaction (DDI study recommended when ratio ≥0.02). Inhibition of 2B6, 2C9, and 3A4/5 was weak (IC50 > 30 µM, representing > 15× Cmax). Rodatristat ethyl and rodatristat are not time-dependent CYP inhibitors, nor inducers of CYP1A2 in hepatocytes. Rodatristat ethyl and rodatristat are weak in vitro inducers of 2B6 (mean induction 37% of control; range, 19–60% and 30%; 8–57%, respectively) and CYP3A4 (10%; 5–13% and 12%; 4–22%) at 10 µM (≥40× Cmax for rodatristat ethyl and ≥5× for rodatristat). Monographs of PAH medications indicate CYP2B6 is not a key metabolizing enzyme. Furthermore, induction of CYP3A is low and unlikely to warrant dose adjustments. In conclusion, most approved PAH medications are metabolized primarily by CYP3A or CYP2C9 and CYP2C8 for treprostinil and selexipag. These isoforms have little role in metabolism of rodatristat ethyl or rodatristat. Rodatristat is metabolically stable and unlikely to be impacted by CYP inhibitors. Although low risk, clinical DDI studies with select PAH medications are planned to validate these predictions.

Investigating the role of the chloride sensing enzyme WNK1 in right ventricle dysfunction in pulmonary arterial hypertension

Sasha Prisco, Megan Eklund and Kurt Prins

Cardiovascular Division, University of Minnesota, Minneapolis, MN, USA

Pulmonary arterial hypertension (PAH) is caused by obstructive remodeling of the pulmonary arteries which ultimately causes right ventricle (RV) failure and death. Increased expression of the glucose membrane transporters, Glut1 and Glut4, occurs in the failing RV, but the upstream regulators of these proteins are unknown. The With-no-Lysine (WNK) kinase-1, a kinase activated in low chloride conditions, promotes expression of Glut1/4 in skeletal muscle, but its regulation in the RV in PAH is unexplored. Furthermore, the relationship between WNK1 and protein O-GlcNAcylation (POGN), a glucose-based post translational modification, has not been examined. Immunoblots quantified the abundance of WNK1, Glut1, Glut4, and total POGN in the RV of control, monocrotaline (MCT) rats, and MCT rats treated with a WNK inhibitor two weeks after MCT injection. Echocardiography assessed RV function and pulmonary artery acceleration time (PA-AT). Results showed that WNK1 expression was increased 1.7 ± 0.4 in MCT RVs. WNK1 expression was significantly correlated with Glut1 (r = 0.86, p = 0.006), Glut4 (r = 0.94, p = 0.0006), and POGN in RV (r = 0.7, p = 0.04) extracts. MCT rats treated with WNK inhibitor had improved TAPSE (2.4 ± 0.1 vs. 1.8 ± 0.2 mm, p = 0.01), cardiac output (80.6 ± 14.7 vs. 48.1 ± 6.8 ml/min, p = 0.07), cardiac output normalized body weight (0.24 ± 0.04 vs. 0.15 ± 0.02 ml/min/g, p = 0.04), and slightly higher PA-AT (18.0 ± 2.6 vs. 13.8 ± 0.7 s, p = 0.17) compared to MCT. At the molecular level, WNK1 inhibition decreased expression of Glut1 (MCT: 2.7 ± 0.9-fold increase, MCT-WNK: 1.2 ± 0.2-fold increase), Glut4 (MCT: 1.7 ± 0.4-fold increase, MCT-WNK: 1.0 ± 0.2-fold increase), and total POGN (MCT: 2.4 ± 0.7-fold increase, MCT-WNK: 1.1 ± 0.1-fold increase). In conclusion, WNK1 expression is increased in RV pressure overload and WNK1 expression is correlated with Glut1/4 and total POGN. WNK inhibition decreased Glut1/4 and total POGN levels, and improved RV function. These results suggest WNK1 promotes RV dysfunction via glucotoxicity and that it may serve as a therapeutic target.

A novel pulmonary epithelial prostacyclin pathway provides systemic anti-thrombotic protection

Nicholas S. Kirkby, Fisnik Shala, Youssef Elghazouli, Harvey R. Herschman and Jane A. Mitchell

National Heart & Lung Institute, Imperial College London, London, UK

Prostacyclin is a powerful anti-thrombotic and vasodilator hormone synthesised by cyclo-oxygenase (COX)-1 enzymes. It is abundantly produced by the lung and isolated vessels where COX-1 and prostacyclin synthase are enriched in endothelial cells (EC). This has led to the universal assumption that the endothelium is the primary source of prostacyclin and been the basis of our understanding of prostacyclin’s role in systemic and pulmonary vascular disease. Here, we have used novel conditional knockout mice where COX-1 is deleted from EC to definitively determine their contribution to pulmonary and vascular prostacyclin production. EC-specific or global COX-1 deletion reduced prostacyclin release from aorta and pulmonary arteries by > 80%. However, in lung parenchyma, total prostacyclin release was reduced by global but not EC-specific COX-1 deletion. Prostacyclin production by aortic EC isolated by fluorescence-activated cell sorting was ∼10-fold greater than by EC isolated from lung parenchyma. Instead, in the lung, the major prostacyclin-producing cells were type I epithelial cells with little contribution from platelets, leucocytes, or other stromal cells. In agreement, type I epithelial cells expressed mRNA for both COX-1 and prostacyclin synthase. To identify a functional role of epithelial-derived prostacyclin, a ferric chloride carotid thrombosis model was used. In EC-specific COX-1 knockout mice, thrombotic occlusion time was reduced by a global COX-1 inhibitor (SC-560) indicating that COX-1 outside of EC actively works to reduce systemic thrombotic tone. These data demonstrate that whilst EC produce the majority of prostacyclin in large systemic and pulmonary blood vessels, within the lung parenchyma, a novel epithelial cell COX-1 pathway is the major the source of prostacyclin and functions to reduce systemic thrombotic tone. Further work is now required to the role of epithelium-derived prostacyclin in pulmonary vascular disease pathology and in cardiovascular consequences of pulmonary airway disease.

Quantitative pulmonary vascular imaging in animal models: a feasibility study

Raúl S.J. Estépar, Pietro Nardelli, George R. Washko and Farbod N. Rahaghi

Division of Pulmonary and Critical Care, Departments of Radiology, Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

Animal models provide unique in-vivo insight for pathophysiology understanding and pharmacological development. The automatic assessment of the pulmonary vasculature in animal models has not been widely demonstrated. Variable imaging quality and anatomy size and variability across species complicate the development of general methods and the automated analysis. Measures of blood volume using scale–space particles have been shown in humans to function as a potential biomarker for pulmonary vascular disease. Here, we will explore the utility of our quantitative pulmonary vascular platform in animal models. Pulmonary vasculature was extracted using the scale–space particle approach that we have developed and used extensively in human epidemiological and clinical studies. Briefly, candidate vascular locations were detected using a deep neural network filter to initialize the scale–space particle algorithm. Scale–space particles were used to grow a topology-free cloud of equally distance points loosely coupled across space and scale arrange along the vascular tree centerline. Scale–space particles were tailored to accommodate the scale and density contrast specific to each animal model and imaging protocol. Vascular size for each particle was regressed using a deep learning approach based on a synthetic vessel model. Quantitative analysis was performed by assessing the distribution of vascular volume as a function of the vascular cross-section area. CT images for a sheep and a mouse were acquired. The image resolution for the sheep and the mouse was 0.6 mm³ and 0.1 mm³, respectively. In both models, large and small intraparenchymal vessels were detected (Fig. 1). The total blood volume was 59.781 mL and 0.768 mL for the mouse and the sheep, respectively. In conclusion, quantitative pulmonary vascular imaging is feasible in large and small animal models using scale–space particles. This study demonstrates the versatility of the geometric approach inherent in the scale–space particle approach to be used in different species.

Fig. 1.

Example of a 3D vascular reconstruction in a patient without chronic thromboembolic disease (a) and one with CTEPH (b) showing loss of small vascular volume (red) in comparison to large vessels (yellow-green-blue). Characteristic values derived from voxel values from dual energy CT imagine as well as the vasculature in the three different cohorts studied (c). Correlations between median voxel value (d), standard deviation of voxel values €, and venous BV10/TBV (f) with pulmonary vascular resistance.

The effect of inhaled treprostinil in the randomized, double-blind, placebo-controlled, phase-III BEAT study

S. Bartolome, R.C. Bourge, H.J. Ford, B. Medarov, J. Sager, S. Shapiro, A.B. Waxman, D. Ishizawar, R. Oudiz, R. Naeije, M. Broderick, E. Borg, A. Nelsen, Y. Rao, C.Q. Deng, P. Sista, Marino M. Di and M.D. McGoon

University of Texas Southwestern Medical Center, Dallas, TX, USA; University of Alabama at Birmingham, Birmingham, AL, USA; University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Albany Medical College, Albany, NY, USA; Cottage PH Center, Santa Barbara, CA, USA; Ronald Reagan UCLA Medical Center, Los Angeles, CA; Brigham & Women’s Hospital, Boston, MA, USA; Medical College of Wisconsin, Milwaukee, WI, USA; David Geffen School of Medicine at UCLA, Los Angeles, CA, USA; University Hospital Brussels, Jette, Belgium; United Therapeutics, Research Triangle Park, Durham, NC, USA; Lung Biotechnology, Silver Spring, MD, USA; Mayo Clinic, Rochester, MN, USA

The event-driven BEAT study evaluated the addition of esuberaprost compared to placebo in patients receiving inhaled treprostinil (ITRE) alone or in combination with one or two additional PAH therapies. Addition of esuberaprost did not demonstrate a clinically significant benefit in delaying time to clinical worsening (CW). The purpose of this exploratory analysis is to further evaluate the effect of ITRE on endpoints in the BEAT study. Patients were experienced or naïve to ITRE at screening. Naïve patients were treated with ITRE for 90-days prior to randomization. Patients were randomized to esuberaprost or placebo, stratified by ITRE experience. CW, exercise capacity (measured by six-minute walk distance (6MWD)), and N-terminal pro-brain natriuretic peptide (NT-proBNP) were assessed. Mixed model repeated measurement was performed to estimate mean treatment change in 6MWD from screening and median ratio of NT-proBNP values to screening (ratio < 1 indicates a decrease). A total of 271 functional class III/IV patients were included in the analysis: n = 73 ITRE-naïve/placebo, n = 78 ITRE-naïve/esuberaprost, n = 62 ITRE-experienced/placebo, and n = 58 ITRE-experienced/esuberaprost. Baseline 6MWD and NT-proBNP were not statistically different between groups. Median duration of ITRE treatment prior to randomization in experienced patients was 375 days; 31.37% of patients achieved > 9 breaths/session ITRE at WK24. Time from screening to CW was statistically different when comparing ITRE-experienced versus ITRE-naïve patients (log rank p = 0.0469; HR (95% CI) 0.673 (0.454–0.997), p = 0.0483). Mean 6MWD changes at WK24 were 39.36 m ITRE-naïve/placebo, 25.01 m ITRE-naïve/esuberaprost, 11.16 m ITRE-experienced/placebo, and 19.12 m ITRE-experienced/esuberaprost. Median NT-proBNP WK24 to screening ratio were 0.65 ITRE-naïve/placebo, 0.66 ITRE-naïve/esuberaprost, 1.09 ITRE-experienced/placebo, and 1.05 ITRE-experienced/esuberaprost. Patients newly initiated on ITRE demonstrated a more robust response than experienced patients. Improvements resulting from ITRE in all groups overshadow any comparison of esuberaprost to placebo, further supporting evidence of the long-term safety and efficacy of ITRE.

Hypobaric hypoxia influences pulmonary hypertension in: frequency, pathogenesis, diagnostic approach and treatment; however, there is very few information on PPHN in altitude

Gabriel F. Díaz, Alicia Márquez, Carlos Sánchez, Luis Ponce, Sergio Torres, Cristian Vargas, Óscar Arévalo and Alberto Olivella

Universidad Nacional de Colombia, Bogota, Colombia; Fundación Santa Fe De Bogota, Bogota, Colombia; Cllínica De La Mujer, Bogota, Colombia; CPO, Valle de Lili, Colombia; Clínica Palermo, Bogota, Colombia; HOMI, Bogota, Colombia

Hypobaric Hypoxia influences pulmonary hypertension in frequency, pathogenesis, diagnostic approach, and treatment; however, there is very few information on persistent pulmonary hypertension of the newborn (PPHN) in altitude. The objective of this study was to analyze patients with PPHN in three maternities of Bogota (2640 MOSL) in the last two years, emphasizing the evolution of patients with early diagnosis. Data were obtained from medical records and echocardiography reports of three institutions. Patients with PPHN were filtered. Demographic data, brain natriuretic peptide (BNP), treatments, and clinical evolution were analyzed. The data were analyzed with statistical packages. Results showed that majority of patients (42 from 46) without early diagnosis, needed mechanical ventilation, high frequency oscillatory ventilation (HFOV), Sildenafil, NO plus Sildenafil-HFOV, and one patient needed extracorporeal membrane oxygenation (ECMO) (2%). Four patients (8.6%) with early diagnosis based on clinical findings: mild polypnea, heart hyperactivity, and loud second heart sound, supported with BNP value, needed only hyperoxia, with resolution of severe PPHN in 24–48 h. Previous studies have demonstrated in newborn rats exposed to hypoxia that muscle cells are developed in distal portions of arterioles from pericytes and intermediate cells. These studies confirm the importance of early detection of PPHN. Analyzing our newborn patients with severe PPHN in last two years in altitude, we can see that the early diagnosis (clinical findings plus echocardiogram) is very important. In these cases, the answer to hyperoxia was very fast, compared with all other patients that needed other therapies and long hospitalization. Other considerations are high frequency of PPHN in altitude and the good correlation of BNP values with the clinical evolution of the patients. In conclusion, early detection of PPHN impact positively the patient evolution (clinical findings are very important). Altitude by hypobaric hypoxia influences frequency and maybe severity of PPHN. BNP is a good marker for diagnosis and follow in PPHN.

Unilateral pulmonary hypertension in a rare congenital heart disease diagnosis and later treatment

Susan Pumacayo-Cárdenas, Moisés Jimenez-Santos, Jose Garcia-Montes and Edgar Quea-Pinto

Departments of Cardiac Tomography, Interventional Cardiology of Congenital Heart Disease, National Institute of Cardiology Ignacio Chávez, México City, México

The incidence of anomalous origin of one pulmonary artery from the ascending aorta (AAo) is 0.1% of all CHD, requiring immediate surgical correction to avoid progressive pulmonary hypertension and congestive heart failure. A 43-year-old male patient, who at nine years of age was diagnosed with patent ductus arteriosus (PDA) and pulmonary hypertension, the PDA was surgically closed. At 34 years of age, he presented hemoptysis. At 43 years of age, he has another episode of hemoptysis and moderate chest pain. A tomography was performed, and an anomalous origin of the right pulmonary artery (RPA) from AAo (AORPA) and bronchiectasis in the right lung was found. The echocardiography showed normal size of left and right ventricles with normal systolic function of both ventricles and AORPA. Cardiac catheterization reported systolic/diastolic pressure of left pulmonary artery (LPA) was 50/25 mmHg (mean 33), systolic/diastolic pressure of RPA was 120/60 mmHg (mean 80), same as systemic pressure, an oxygen vasoreactivity test (OVT) was performed without significant variation. Surgical implantation of the RPA to the main pulmonary artery (MPA) with a 16 mm diameter woven Dacron tube was decided. One month after surgery, a heart catheterization was performed that concludes: pulmonary pressure (PP) of 119/49 mmHg (mean: 77 mmHg), PVR 18.7 UWood/m², 100% OVT was performed and a decrease in PP and PVR was found (68/19 mmHg, mean of 41, and PVR 7.8 UWood/m²). Twenty months after surgery, LPA CT and catheterization was performed and found a mean PP of 33 mmHg and PVR 12 Uwoods/m², the clinical evolution is favorable, and sildenafil was received. Some authors suggest that surgical correction should be performed during the first three months of life. We report the case of an older patient of all series undergoing surgical correction of AORPA.

Fig. 1.

Example of a 3D iodine map with a large perfusion defect (a), subsequent imaging shows resolution of the perfusion defect with decrease in the standard deviation of the iodine map from 78 to 35 (b). Vascular reconstruction from the same patient (c) with increase in small vessel volume (red/orange) on the subsequent scan (d). Correlation between venous small vessel fraction and standard deviation of iodine map values across the cohort using the arterial € and the venous (f) circulation.

Fig. 1.

CT images, vascular reconstructions and vascular volume distribution profiles for two animal models. (Top row): Sheep model and (bottom row) mouse model. The vascular profiles represent the distribution of vascular volume per vessel cross sectional area.

Pulmonary banding, solution or problem in a third level hospital, Peru

Susan Pumacayo-Cárdenas and Edgar Quea-Pinto

Department of Specialized Pediatrics, Edgardo Rebagliati Martins National Hospital, Lima, Perú

In our country, there are no studies on the evaluation of pulmonary artery banding (PAB) in children with congenital heart disease and pulmonary hyperflow. The objective of this study was to determine PAB results in patients with congenital heart disease and pulmonary hyperflow in a third level hospital, Lima, Perú. Review of clinical records of patients operated on PAB, who met the selection criteria, was performed. Descriptive statistics were used. There were 38 pulmonary banding procedures. The most frequent female sex (63%), from Lima (84%), 94% were RNAT and PAEG, 47% had Down syndrome, and 71% had some genetic syndrome. The most frequent heart disease was CIV plus PCA (52.6%). The most frequent age was 1–6 months (52.6%). The most frequent weight was 3500 g. The most frequent preoperative comorbidity was sepsis (26%). The most frequent surgical technique was the usual > 90% of the cases and in 21% surgical closure of PCA was also performed. The most frequent O² saturation was 83–96% and the average transbanding gradient was 56 mmHg. The most frequent postoperative clinical features were: inadequate nutritional recovery 21.1%, persistent ICC (first six months) 44.7%, and functional class improvement in 36.8%. The most frequent intraoperative complication was hypotension plus severe bradycardia. The most frequent early postoperative complication was congestive heart failure (CHF) 50%. The most frequent late complication was pulmonary trunk dilatation (47.4%). The postoperative stay in ICU was 24 days, average. Mortality was 36.84%. The mean waiting time for the BAP was 3.9 months. The reference waiting time for corrective surgery was 18.8 months. The average waiting time for corrective surgery was six months. BAP is a procedure prior to corrective surgery that requires an adequate selection of patients, our study has shown improvement of the patients in the postoperative period; however, it is necessary to decrease the high risk of mortality and shorten the reference for corrective surgery.

Discovery of transverse airway–vessel interactions in the bronchovascular bundle: implications for therapeutic innovation in pulmonary vascular disease

Jane A. Mitchell, Abel Tesfai, Renate Paddenberg, Petra Mermer, Ricky Vaja, Blerina Ahmet-Shala and Nicholas S. Kirkby

Cardiothoracic Pharmacology, National Heart and Lung Institute, Imperial College London, London, UK; Institute of Anatomy and Cell Biology, Giessen, Germany

There are few remaining structures within our bodies for which we know nothing about their function or role in disease; the bronchovascular bundle is one of them. The bronchovascular bundle is made up of a single structure that contains the airways and pulmonary arteries in a defined sheath within the lungs. Despite the airway and vessel being tightly bound together in one biological complex structure, physiological functions, and diseases of the airways, such as asthma and of pulmonary arteries, such as pulmonary arterial hypertension, are generally studied separately. This has meant that previous experiments have involved cutting out either the airway or the vessel completely disregarding the importance of the bronchovascular bundle. Appreciating the functional role of the bronchovascular bundle would initiate an entirely new area of research and could be absolutely critical in defining a new understanding of heart and lung function in health and disease. Here we have used imagining of precision cut living lung slices to report a new phenomenon akin to a ‘physiological pull’ of pulmonary arteries towards a contracting airway and RNA sequencing to demonstrate site-specific gene expression in airways and vessels dependent on their proximity to each other within the bronchovascular bundle. Importantly others have used similar lung slice and imagining techniques in identical settings to our work. In those studies, selective contraction of the airway also clearly resulted in the tensile pull we report here but, in those studies, to our knowledge, the phenomenon was not quantified, acknowledged or specifically discussed. In conclusion, our observations define a completely new paradigm within which a functional role of the bronchovascular bundle must now be considered. Our study provides the impetus for future work to determine how communication between the structures occurs, the signalling pathways involved and the identity of novel anti-inflammatory checkpoints.

Artificial intelligence highlights novel risk factor in pulmonary endarterectomy

Katherine Bunclark^*, James Liley^*, Michael Newnham, Alessandro Ruggiero, John E. Cannon, Gerry Coghlan, James Lordan, Luke Howard, David Jenkins, Martin Johnson, David G. Kiely, Choo Ng, Nicholas Screaton, Karen Sheares, Dolores Taboada, Steven Tsui, S.J. Wort, Joanna Pepke-Zaba and Mark Toshner

Royal Papworth Hospital, University of Cambridge, Cambridge, UK; University of Birmingham, Birmingham, UK; Royal Free Hospital, London, UK; Institute of Cellular Medicine, Newcastle Upon Tyne, UK; National Heart and Lung Institute, London, UK; Golden Jubilee National Hospital, Glasgow, Scotland; Sheffield Teaching Hospitals, Sheffield, UK; Royal Brompton Pulmonary Hypertension and Adult Congenital Heart Centre, London, UK

^*These first authors contributed equally.

Despite improving survival rates following pulmonary endarterectomy (PEA), there remains an unavoidable mortality risk even in the best centres. Although clinical risk prediction tools are in use for other cardiothoracic surgeries, no systematic method for estimating individualised PEA risk presently exists. The objectives of this study were to identify pre-operative risk factors of early (90-day) and late (five-year) mortality following PEA for inclusion in a clinically-implementable risk prediction tool. Consecutive chronic thromboembolic pulmonary hypertension (CTEPH) patients undergoing PEA at Royal Papworth Hospital, UK, from 2007 to 2017 were included. Potential pre-operative predictors including patient demographics, medical history and results of functional, physiological and patient self-reported measures were included in a hypothesis-free approach. Three statistical predictive models were considered (linear regression, lasso regression and random forest), each of which were calibrated, fitted and assessed using cross-validation ensuring internal consistency. Results showed that of the 1336 individuals included in risk modelling, 96 (6.4%) died within 90 days and 154 (11.5%) within five-years of PEA. The strongest predictor model was random forest. Early (AUROC 0.82: 95%CI: 0.78–0.87) and late (C-Index: 0.81: 95%CI: 0.76–0.85) mortality were both predicted well from pre-operative non-invasive variables. PEA-specific models out-performed standard generic cardiothoracic prediction tools (EUROscore II: AUROC 0.72, p < 1 × 10⁻⁵). Left atrial dilatation was the strongest individual risk factor for both early and late mortality. Adverse functional, haemodynamic and patient-reported outcomes were also seen in this group who were enriched for signs of left ventricular hypertrophy and diastolic dysfunction. In conclusion, early and late mortality risk from PEA can be predicted to a clinically useful degree from pre-operative non-invasive variables to enable the generation of a PEA-specific risk calculation tool. Whilst left ventricular diastolic dysfunction has been implicated as an adverse prognostic marker in other cardiothoracic surgeries, this is the first demonstration of its potential importance in CTEPH and PEA outcomes.

Primary results from the randomized, double-blind, placebo-controlled phase 3 trial – Beraprost-314d Added to Tyvaso (BEAT) in World Health Organization (WHO) Functional Class (FC) III and IV patients with pulmonary arterial hypertension (PAH)

H.J. Ford, M. Kramer, S. Bartolome, R. Saggar, R.C. Bourge, B. Medarov, J. Sager, S. Shapiro, A. Waxman, D. Ishizawar, R. Oudiz, R. Naeije, J. Shin, P. Sista, M. Di Marino, A. Smart, M.-L. Tomson and M. Lorber

UNC-CH, Chapel Hill, NC, USA; Rabin Medical Center, Petah Tikva, Israel; UT-SW Medical Center, Dallas, TX, USA; UCLA-David Geffen Medical Center, Los Angeles, CA, USA; UAB, Birmingham, Alabama; Albany Medical College, Albany, NY, USA; Cottage PH Center, Santa Barbara, CA, USA; Brigham & Women’s Hospital, Boston, MA, USA; Medical College of Wisconsin, Wauwatosa, WI, USA; University Hospital Brussels, Jette, Belgium; Lung Biotechnology, Silver Spring, MD, USA

A short half-life of some prostanoid PAH therapies leads to oscillation between peak and trough plasma drug levels, with undesirable consequences of side-effects and symptoms. The BEAT study tested a hypothesis that combination of two prostanoids, esuberaprost (ESU), and inhaled Treprostinil (ITRE), delivered by oral and inhaled routes, respectively, would benefit patients by simulating continuous parenteral drug delivery. BEAT was an enrichment trial for FC III–IV PAH patients at 77 US and Israel sites. All patients received background therapy: ITRE plus oral PAH therapy (endothelin receptor antagonists (ERAs) and/or phosphodiesterase-5 inhibitors (PDE5i)/soluble guanylate cyclase (sGC) stimulators only). Patients were randomized 1:1 to ESU (fixed dose of 28.4 µg four times a day) or PBO. The primary endpoint was time to clinical worsening (TtCW), i.e. from randomization to the first occurrence of any one of the following: death (all causes), hospitalization due to worsening PAH, use of parenteral prostanoid, disease progression, or unsatisfactory long-term clinical response. Secondary endpoints included: six-minute walk distance, Borg dyspnea score, WHO FC, and N-terminal pro-brain natriuretic peptide. A total of 273 patients were randomized; 271 received study drug (136 in ESU and 135 in placebo (PBO) arms, respectively). Demographics and baseline characteristics were similar in both arms. The addition of ESU to ITRE-containing background therapy did not delay TtCW (p = 0.62; Hazard Ratio 0.9 (0.6–1.3)); subgroup analyses did not identify factors favoring ESU. Overall ESU was safe and well tolerated. Despite enrichment with WHO FC III–IV patients, the overall event rate at one-year was 19% as opposed to the hypothesized 35–45% rate. The addition of a second prostanoid in advanced PAH patients did not demonstrate incremental clinical benefit. The lower than expected event rate suggests ITRE contributed to a robust background and perhaps limited discernment of ESU’s pharmacologic activity and potential clinical efficacy in the study.

Videocapillaroscopic changes in patients with Eisenmenger syndrome indicate systemic microangiopathy

Alexandra Arvanitaki, Theodoros Dimitroulas, Eva Triantafyllidou, Christos Feloukidis, Sophia-Anastasia Mouratoglou, Haralambos Karvounis, Alexandros Garyfallos and George Giannakoulas

First Department of Cardiology, AHEPA University Hospital, Thessaloniki, Greece; Fourth Department of Internal Medicine, Hippokration University Hospital, Medical School, Aristotle University of Thessaloniki, Thessaloniki, Greece

Limited amount of evidence suggests the presence of systemic vasculopathy in pulmonary arterial hypertension (PAH), whereas there is hardly any evidence regarding patients with Eisenmenger syndrome. Nailfold videocapillaroscopy (NVC) is an established, non-invasive imaging technique for the assessment of the microcirculation. The aim of this study is to investigate the presence of capillaroscopic abnormalities in patients with the Eisenmenger syndrome. We present preliminary data regarding capillaroscopic abnormalities in patients with Eisenmenger syndrome treated in a PH expert center in Greece. All patients and healthy controls underwent NVC, performed on all fingers of both hands, excluding thumbs, using Optilia Digital Capillaroscope. Capillary quantitative and qualitative parameters were measured. NVC was performed in 15 patients with Eisenmenger syndrome (mean age: 41.5 ± 14.4 years, 66.7% female, 40% with a ventricular septal defect) and 28 healthy controls (mean age 42.5 ± 13.5 years, 71.4% female). Significant capillaroscopic abnormalities were detected in patients compared to healthy controls. Capillary density was significantly decreased in patients with Eisenmenger syndrome vs. healthy controls (8.8 ± 0.9 loops/mm vs. 9.7 ± 0.8 loops/mm, p = 0.002). Patients with Eisenmenger syndrome also presented increased mean capillary width (38.3 ± 6.4 µm vs. 29.5 ± 4.4 µm, p < 0.001), increased loop diameter (16.7 ± 2.8 µm vs. 11.5 ± 2.2 µm, p < 0.001), and increased mean number of irregularly enlarged capillaries > 50 µm/mm (0.5 (IQR: 1.3) vs. 0.2 (IQR: 0.6), p = 0.01). Furthermore, capillary shape abnormalities, microhemorrhages, and signs of thrombosis were more frequent in Eisenmenger patients compared to healthy controls (2.7 ± 1.0 abnormal capillaries/mm vs. 1.2 ± 0.8 abnormal capillaries per mm, p < 0.001, 0.12 (IQR: 0.37) microhemorrhages/mm vs. 0.0 (IQR: 0.0) microhemorrhages per mm, p < 0.001, 2.6 ± 1.4 thrombosed capillaries per mm vs. 1.5 ± 0.7 thrombosed capillaries per mm, p = 0.014]. These data support for the first time the hypothesis of systemic microvascular involvement in Eisenmenger syndrome in addition to the well-known pulmonary vasculopathy.

Age-associated differences in pulmonary hypertension patients

Z. Safdar, R. Bui and S. Rayasam

Houston Methodist Hospital, Weill Cornell College of Medicine, Houston, TX; Rice University, Houston, TX, USA

Pulmonary arterial hypertension (PAH) is historically considered a disease affecting females of childbearing potential; however, recent data suggests that majority of patients are older and postmenopausal. In order to evaluate the age distribution in our cohort, we collected clinical, hemodynamic and biomarker data sorted across tertile age ranges from prevalent PAH patients. Data from 88 PAH patients were collected from the Houston Methodist Pulmonary Hypertension Center. Clinical, hemodynamic, echocardiogram, and six-minute walk data were collected, followed by an analysis of biomarker levels from peripheral blood samples taken once from each patient. All patients were then stratified into tertiles by age (18–40 years, ≤41–54 years, ≤54–78 years). A one-way analysis of variance was used to identify important statistical significances, determined as p-value < 0.05. Age of patients in this cohort was 47 ± 15 years (mean ± SD), and 90% of the patients were female. Duration of disease since diagnosis (p = 0.005), and concurrently use of beta-blockers (p = 0.018) increased with age. There was a trend toward a lower pulse rate with age (p = 0.057). Contrary to published data, in our cohort, N-terminal propeptide of type III procollagen levels decreased with age (p = 0.007). The echocardiogram data suggested that both cardiac output and cardiac index were lower in the younger age group as compared to the older PAH patients (p = 0.019 and 0.020, respectively). Similarly, diffusing capacity of the lung for carbon monoxide decreased with age (p = 0.001). Interestingly, there was no significant difference in mortality across the tertiles. In our cohort, hemodynamics, biomarkers, and echocardiogram data differed across age groups though the overall long-term survival was not different. This data suggested that there may be a survival bias for patients with milder disease. Further studies are needed to investigate these findings.

Development and validation of a multivariable model for five-year survival in systemic lupus erythematosus-associated pulmonary arterial hypertension: CSTAR-PAH cohort study

Jingge Qu, Junyan Qian, Jiuliang Zhao, Qian Wang, Mengtao Li and Xiaofeng Zeng

on behalf of CSTAR-PAH collaborators

Department of Rheumatology, Peking Union Medical College Hospital, Peking Union Medical College & Chinese Academy of Medical Sciences, National Clinical Research Center for Immunologic Diseases, Ministry of Science & Technology, Key Laboratory of Rheumatology and Clinical Immunology, Ministry of Education, Beijing, China

Pulmonary arterial hypertension (PAH) is the major mode of death in systemic lupus erythematosus (SLE), but there is no validated algorithm to identify those at highest risk. A prognostic model was developed from a multicenter, longitudinal cohort study of 310 consecutively evaluated patients with SLE-associated PAH. The study was conducted from November 2006 to January2019. Death was the primary outcome. The model was developed using Cox proportional hazards regression modeling. We developed a prognostic index (PI), summing the number of risk points corresponding to weighted covariates, which were used to configure the nomogram. Internal validation of the nomogram was assessed by discrimination and calibration using bootstrapping. Of the 310 patients included in the study, 68 deaths (22.2%) occurred at a median follow-up of 4.9 (interquartile range: 3.2–6.3) years. The final prognostic model included six variables: N terminal-pro brain natriuretic peptide (NT-proBNP), lactic dehydrogenase, direct bilirubin, six-minute walking distance, serositis, and alopecia. Five-year survival probability-predictive nomogram with PI in the main analysis was established (Fig. 1). The model’s ability to predict risk was validated with C statistic (0.82 (95%CI: 0.73–0.91)) and calibration curve. Risk stratification was made based on PI to improve the primary prevention and management of SLE-associated PAH. This new, validated risk stratification model for SLE-PAH may provide individualized estimates of risk at five years using readily obtained clinical risk factors. External validation studies are required to demonstrate the accuracy of this model’s predictions in diverse patient populations. (This work was supported by the Chinese National Key Technology R&D Program, Ministry of Science and Technology (2017YFC0907601, 2017YFC0907602), and the Chinese National High Technology Research and Development Program, Ministry of Science and Technology (2012AA02A513)).

Fig. 1.

The nomogram based on the six significant predictors for survival.

Gender-related serum gamma-glutamyltransferase in idiopathic pulmonary arterial hypertension

Su-Gang Gong, Chao Li, Gang-Hua Lu, Lan Wang and Rui Zhang

Department of Cardio-Pulmonary Circulation, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China; Tongji University School of Medicine, Shanghai, China

It has been suggested that the elevated gamma-glutamyltransferase (γ-GT) level was an indicator of liver dysfunction and cardiometabolic disorders with high female predominance but low female mortality. The incidence of idiopathic pulmonary arterial hypertension (IPAH) was higher in female, whereas prognosis was poor in male patients. Therefore, we aimed to determine whether γ-GT showed great difference in male and female IPAH patients. Serum γ-GT levels were measured in 300 patients with IPAH from 2010 to 2018 and matched 100 healthy subjects by sex and age. The followed interval was 13 ± 11 months. The median level of serum γ-GT was significantly higher in patients with IPAH than those of control subjects. Baseline γ-GT was increased in male patient compared with those of female patients. By multivariate analysis, it proved that the serum γ-GT was an independent risk factor with mortality. When the end of follow-up time, survivors has decreased level of γ-GT, while the level in the death was increased. Patients with higher cut-off value of baseline γ-GT level had a worse survival rate than those with lower level. Male patients had increasing mortality risk than female patients. In conclusion, the γ-GT had a great sex difference in IPAH patients and served as a prognostic indicator. Increased γ-GT level in male IPAH patients might be a factor of poor prognosis.

Safety and efficacy of combination therapy with ambrisentan and tadalafil for the treatment of pulmonary arterial hypertension in children: early experience

Azadeh Issapour, Sarah Crook, Michelle Hite, Erika B. Rosenzweig, Benjamin Frank, Ivy D. Dunbar and Usha Krishnan

Division of Pediatric Cardiology, Department of Pediatrics, Columbia University Medical Center, New York, NY, USA; Division of Pediatric Cardiology, Children’s Hospital Colorado, Aurora, CO, USA

The AMBITION trial revealed lower risk of clinical failure events with ambrisentan and tadalafil combination as compared to monotherapy alone in adults. We aimed to describe the safety and efficacy of this combination therapy in children with PAH. Retrospective observational study including children from two Pediatric PAH centers was performed. Demographics, symptoms, exercise capacity, hemodynamics, biomarkers, and adverse effects were collected at baseline (before start of ambrisentan and tadalafil) and early and late follow-up. Wilcoxon matched-pairs signed-rank tests were used to test for statistical significance. Patients either switched from a different dual PDE5 + ERA combination (group A) or received no prior PAH therapy or monotherapy (group B). There were 29 subjects, (16 F, 13 M; aged 6.5–13.5 years, median 10) with Group I PAH. All subjects reported improvement in symptoms. Statistically significant improvements in 6MWD and hemodynamics were seen at early and late follow-up (14–23 months, median 19) in the entire sample and Group B (Table 1). WHO FC improved in 41% and was unchanged in 59%. Two discontinued tadalafil due to headaches and none due to lack of clinical efficacy. In conclusion, combination therapy with ambrisentan and tadalafil was associated with improvement in exercise capacity and hemodynamics in children as compared to monotherapy, and had an acceptable safety profile. Based on these early data, further study of combination therapy in pediatric PAH is warranted.

Table 1.

Change scores between baseline and follow-up.

Change score	6MWD (m)	pro-B-type natriuretic peptide (ProBNP)	mPAPa	PVRia	PVRi/systemic vascular resistance index (SVRi)a
All (n = 29)
3–6 months	26.0 (–2.0 to 31.0)b	–53.7 (–193.0 to 67.4)	–6.0 (–11.0 to –1.0)b	–1.7 (–11.7 to 1.0)b	–0.04 (–0.42 to 0.11)
Most recent	37.0 (–14.0 to 86.0)b	–40.5 (–145.3 to 50.0)
Group A (n = 12)
3–6 months	10.0 (–5.0 to 26.5)	5.6 (–47.2 to 103.7)	–6.0 (–7.0 to 3.0)	1.0 (–0.9 to 2.4)	0.05 (–0.36 to 0.28)
Most recent	–14 (–30 to 31)	22.5 (–90.6 to 130.2)
Group B (n = 17)
3–6 months	27.0 (20.0 to 45.0)b	–63.6 (–193.3 to 31.0)	–9.0 (–20.5 to –1.5)b	–-11.3 (–13.5 to –1.5)b	–0.07 (–0.47 to 0.01)
Most recent	71.0 (26.0 to 109.0)b	–60.3 (–160.1 to 3.1)

mPAP, mean pulmonary arterial pressure; PVRi, pulmonary vascular resistance index.

Note: Data are presented as median (IQR).

^aMedian time to first follow-up catheterization was 8.5 months

^bp ≤ 0.05.

Targeting Janus kinase-signal transduction and activator of transcription (JAK-STAT) signaling in experimental pulmonary hypertension

Dinesh R. Yerabolu, Astrid Weiss, Baktybek Kojonazarov, Norbert Weissmann, Ardeschir Ghofrani, Friedrich Grimminger, Werner Seeger and Ralph Schermuly

Universities of Giessen and Marburg Lung Center (UGMLC), Giessen, Germany; Member of the German Center for Lung Research (DZL), Giessen, Germany

Lung vascular remodeling is the hallmark of pulmonary arterial hypertension (PAH) and attributed to the increase of proliferation and resistance to apoptosis of pulmonary vascular cells. A number of proliferative mediators including platelet-derived growth factor and cytokines are thought to play an important role in the pathogenesis of the abnormal phenotype of the pulmonary vascular cell in PAH. Amongst others, they can activate the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway. Deciphering the molecular details of aberrant signaling in pulmonary vascular cells in pulmonary (arterial) hypertension is fundamental for the development of new therapeutic strategies. Therefore, an effective strategy using potent small molecule inhibitors like ruxolitinib should target ligand-induced receptor tyrosine kinases and their downstream signaling pathways like JAK-STAT to interfere with cellular and structural alterations of the pulmonary vasculature. The aim of our study is to identify tyrosine kinases that are dysregulated in PAH by means of a kinase activity assay. We performed ex vivo multiplex tyrosine phosphorylation assays using 144 selected micro arrayed kinase substrates and analyzed tyrosine kinase activities in pulmonary arterial smooth muscle cells from PAH patients. In vitro, proliferation and migration were studied and in vivo experiments were performed in two experimental models of pulmonary hypertension. We observed an over-activation of Janus kinase-2 (Jak2) activity in pulmonary arterial smooth muscle cells from PAH patients. The Jak2 inhibitor ruxolitinib inhibited interleukin-6 stimulated pulmonary arterial smooth muscle cell proliferation and migration. In vivo, ruxolitinib dose-dependently attenuated elevated pulmonary arterial pressure, reduced right heart hypertrophy, and restored cardiac index in hypoxia-induced PH in mice and monocrotaline-induced PH in rats. Here, we conclude that ruxolitinib is a novel therapeutic agent in PAH by effectively blocking Jak2-Stat3-mediated signaling pathways.

International environmental scan of specialized pulmonary hypertension clinics

Carolyn Doyle-Cox, Gail Nicholson, Traci Stewart and Wendy Gin-Sing

University of Ottawa Heart Institute, Ontario, Canada; Alberta Health Services, Alberta, Canada; Heart and Vascular Center, University of Iowa, Iowa City, IA, USA; Hammersmith Hospital, Imperial College Healthcare NHS Trust, London, UK

Pulmonary arterial hypertension (PAH), a subcategory of pulmonary hypertension (PH), is a rare and progressive disease with no known cure. Despite recent advances in therapy, the five-year mortality of patients with PAH remains as high as 40%. Disease management through multi-disciplinary community care clinics has been shown to improve patient outcomes in many health conditions, i.e. heart failure. The potential benefits of a multi-disciplinary strategy include improved utilization and compliance with evidence-based therapies, which may lead to increased length and quality of life in people living with PAH. The objectives of this study were to assess the range of multi-disciplinary services provided through specialized PH clinics at the international level. A team of four PH nurses from Canada, the USA and the UK and a PH patient living in Canada collaborated to create a survey to capture information on clinic setup, patient demographics, specialty physician and nurse availability, treatment protocols, research involvement, and collaborative care in pulmonary hypertension clinics around the world. Thirty-five questions were available in English, French, Spanish, Russian, Farsi, Korean, Italian, German, and simplified Chinese. Centers were identified by the PH Associations in each country and were e-mailed the link to the questionnaire detailing the purpose of the study. The information was collected from over 100 clinics worldwide. Over 126 responses were collected across more than 32 countries. The gathered information will be presented and discussion around a more targeted approach for creating and sustaining multidisciplinary centers of excellence for optimal treatment and management of PAH. The survey is the beginning of a larger project. The next steps will be to create a template that identifies a PH center of excellence with patient outcomes and quality of life being the measuring sticks. Centers that meet all future decided upon criteria will eventually be accredited internationally.

A multi-layer microfluidic device to recapitulate pediatric pulmonary arterial hypertension (PAH) for studying pediatric PAH pathophysiology and developing age-specific therapy

Hossam Kadry and Fakhrul Ahsan

Department of Pharmaceutical Sciences, Texas Tech University Health Sciences Center, Amarillo, TX, USA

Pediatric pulmonary arterial hypertension (PAH) affects 2–16 children per million of the pediatric population. Although the etiology and progression of pediatric PAH differ from those of adult PAH, the therapy for pediatric patients is still based on the clinical outcome from adult patients. Currently, PAH investigators use young animal and two-dimensional cellular models to investigate the pathophysiology of PAH in children and to study the efficacy of anti-PAH drugs. However, these models cannot accurately recapitulate the disease, nor do they allow simulation of three-dimensional tissue-level function. We propose here to deploy the microfluidic technology to test the hypothesis that a pediatric-PAH-on-a-chip can imitate the pathophysiology of PAH in children, and thus help illuminate pediatric PAH pathogenesis and develop therapies tailored to patient age. Here, we present a chip device mimicking five layers of a pulmonary artery (perivascular, adventitial, medial, intimal, and luminal) and showed that our five-channel device can reproduce various pathologies of pediatric PAH (Fig. 1). Using this cell-laden device, we simulated vessel muscularization, a chief pathologic feature of PAH-afflicted pulmonary arteries, by growing PAH-endothelial cells (ECs) along with PAH-smooth muscle cells (SMCs) plus PAH-adventitial cells (ADCs) and normal (N)-ECs along with normal (N)-SMCs plus normal (N)-ADCs as parallel controls. Laminin and collagen, two major extracellular matrix proteins, deposition was observed in chips prepared with PAH cells, but not in chips with normal cells. PAH-SMCs appeared to invade PAH-ECs, a process resembling muscularization. PAH-ECs, when grown in the presence of PAH-SMCs plus PAH-ADCs, showed an aggressive proliferation pattern in the luminal layer. PAH-ECs expressed fibroblast specific protein-1 (FSP-1) and smooth muscle alpha-actin (α-SMA), which N-ECs do not normally express, and these transdifferentiated ECs migrated from the intimal to the luminal layer. Overall, the above studies demonstrate the feasibility of reproducing the major pathologies of pediatric PAH in our chip-based model.

Fig. 1.

(a) A linear chip comprising five channels mimicking the adventitial, medial, and intimal layers for growing adventitial (ADCs), smooth muscle (SMCs), and endothelial cells (ECs), respectively, and perivascular and luminal layers for the delivery of growth media or treatments. Each channel is 1 min wide and 3 cm long, separated by 150-µm-tall trapezoidal pillars placed 200 µm apart. (b) Photographs of chips fabricated by using the soft lithography technique. After fabrication and attachment of the chips to glass slides, the chips were tested for any leakage by passing trypan-blue solution through the channels. Because of the laminar flow, the growth medium or cell suspension creates a static interfacial film between the pillars. The resulting film keeps the medium or cell suspension confined within the same channel.

Characterizing patients treated with macitentan: real-world evidence and a randomized controlled trial cohort

Nick Kim, Kelly Chin, Vallerie McLaughlin, Cassandra Lickert, Michele Cole, Megan Flynn, Carol Zhao and Richard Channick

University of California, San Diego, La Jolla, CA, USA; UT Southwestern Medical Center, Dallas, TX, USA; University of Michigan, Ann Arbor, MI, USA; Actelion Pharmaceuticals US, Inc, South San Francisco, CA, USA; University of California Los Angeles, Los Angeles, CA, USA

Randomized controlled trial (RCT) data are considered gold standard for evaluating safety and efficacy of interventions but may not completely reflect real-world clinical practice. We aim to describe characteristics, treatment patterns, safety, and outcomes of pulmonary arterial hypertension (PAH) patients treated with macitentan in an RCT and real-world clinical practice. SERAPHIN (NCT00660179) was a double-blind, placebo-controlled, event-driven, phase III study to assess effect of macitentan in PAH. OPUS (NCT02126943) is an ongoing, long-term, prospective, observational drug registry of patients newly treated with macitentan 10 mg. SERAPHIN enrolled 742 PAH patients; 242 (32.6%) were randomized to macitentan 10 mg. OPUS enrolled 1710 PAH patients with follow-up data (as of October 2018). Compared to the 10 mg macitentan arm of SERAPHIN, patients in OPUS were older (mean age: 60.4 vs. 45.5 years), had fewer WHO FC (I/II) patients (28.6 vs. 50.0%), fewer on background PAH therapy (59.4 vs. 63.6%), and a higher proportion of patients with hypertension (52.9 vs. 35.5%), diabetes (23.7 vs. 5.4%), or edema (25.6 vs. 5.8%) at baseline (Table 1). The mean six-minute walk distance was shorter in OPUS (42% reported) (298.4 ± 132.2 m) vs. SERAPHIN 363 ± 93.2 m). The comparison of adverse events (AEs) indicate that fewer OPUS patients experienced peripheral edema (9.4 vs. 18.2%) and the number of PH-related hospitalizations per year was 0.3 hospitalization/year for both). The discontinuation of macitentan due to AE (including hepatic AE) was more frequent for the OPUS patients (18.3 vs. 10.7%). SERAPHIN is the largest RCT assessing safety and efficacy of an endothelin receptor antagonists (ERA). OPUS is the largest real-world cohort of newly treated ERA patients. Patients enrolled in OPUS were older, had more baseline co-morbidities, lower rates of edema as an AE, and fewer on PAH background therapy; regardless of these differences, hospitalization was the same.

Table 1.

Summary of disease characteristics, medical history and macitentan exposure.

	SERAPHIN macitentan 10 mg (n = 242)	OPUS (n = 1710)
Characteristic at time of initiation
Female sex, n (%)	194 (80.2)	1273 (74.4)
Age, year (mean ± SD)	45.5 ± 14.99	60.4 ± 14.49
Race, n (%)
White	135 (55.8)	1321 (77.3)
Black	6 (2.5)	264 (15.4)
Asian	65 (26.9)	N/A
Other	36 (14.9)	125 (7.3)
Time from diagnosis of PAH, year	2.6 ± 3.6	2.5 ± 4.9
PAH classification, n (%)
Idiopathic	134 (55.6)	979 (57.3)
Associated with connective-tissue disease	73 (30.3)	444 (26.0)
Associated with congenital shunts	21 (8.7)	88 (5.1)a
Heritable	2 (0.8)	30 (1.8)
Other	11 (4.6)	169 (9.9)
Six-minute walk distance, m	363 ± 93.2	298.4 ± 132.2; n = 718
WHO functional class, n (%)
I	1 (0.4)	128 (7.5)
II	120 (49.6)	361 (21.1)
III	116 (47.9)	683 (39.9)
IV	5 (2.1)	77 (4.5)
Missing	0	461 (27.0)
Medical history (reported in $\ge$ 10% of patients)
Hypertension	86 (35.5)	905 (52.9)
Diabetes mellitus	14 (5.4)	405 (23.7)
Edema (oedema peripheral in SERAPHIN)	13 (5.8)	437 (25.6)
Anemia	13 (5.4)	291 (17.0)
Signs of right heart failure (right ventricular failure in SERAPHIN)	83 (34.3)	275 (16.1)
Renal insufficiency	0	215 (12.6)
Autoimmune disease	0	221 (12.9)
Gastroesophageal reflux disease	27 (11.2)	14 (0.8)
Receipt of background treatment for PAH, n (%)
Yes	154 (63.6)	1016 (59.4)
No	88 (36.4)	694 (31.6)
Macitentan treatment duration, months	23.9 ± 12.1b	15.2 ± 13.0

PAH, pulmonary arterial hypertension; N/A, not available.

Notes: For continuous variables, means ± SD were presented. In OPUS, missing values exist. n was added to indicate the sample size the result was based upon. Results for SERAPHIN were from NEJM Pulido 2013 paper.

^aOPUS includes all congenital heart diseases.

^bNot reported in the reference paper.

The impact of time from diagnosis at baseline on long-term outcome in the GRIPHON study: selexipag in pulmonary arterial hypertension (PAH)

Sean Gaine, Olivier Sitbon, Richard N. Channick, Kelly M. Chin, Lilla Di Scala, Nazzareno Galiè, Marius M. Hoeper, Vallerie V. McLaughlin, Ralph Preiss, Lewis J. Rubin, Gérald Simonneau, Victor Tapson, Hossein-Ardeschir Ghofrani and Irene Lang

National Pulmonary Hypertension Unit, Mater Misericordiae University Hospital, Dublin, Ireland; Hôpital Universitaire de Bicêtre, Université Paris-Sud, Le Kremlin Bicêtre, France; UCLA, Los Angeles, CA, USA; UT Southwestern Medical Center, Dallas, TX, USA; Actelion Pharmaceuticals Ltd, Allschwil, Switzerland; Department of Experimental, Diagnostic and Specialty Medicine – DIMES, University of Bologna, Bologna, Italy; Department of Respiratory Medicine, Hannover Medical School and German Center of Lung Research, Hannover, Germany; Division of Cardiovascular Medicine, Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA; Division of Pulmonary and Critical Care Medicine, University of California, San Diego, CA, USA; Cedars-Sinai Medical Center, Los Angeles, CA, USA; University of Giessen and Marburg Lung Center, Giessen, Germany; German Center of Lung Research, Department of Medicine, Imperial College London, London, UK; Division of Cardiology, Department of Internal Medicine II, Allgemeines Krankenhaus, Medical University of Vienna, Vienna, Austria

Newly diagnosed PAH patients have a poor prognosis. In the randomized, event-driven, long-term GRIPHON trial, the oral IP prostacyclin receptor agonist selexipag significantly reduced the risk of morbidity/mortality events compared with placebo in PAH patients. This post-hoc analysis explores whether early initiation of selexipag, with regards to time from diagnosis, improves outcomes for PAH patients. The treatment effect of selexipag versus placebo on the primary endpoint (composite morbidity/mortality) was evaluated for patients categorized based on their time from diagnosis at baseline using a six-month threshold to define the newly diagnosed patients: patients treated earlier (time from diagnosis ≤6 months) and later (time from diagnosis > 6 months). Kaplan–Meier estimates by treatment arm and subgroup were calculated. Hazard ratios (HRs) with 95% confidence intervals (CIs) were calculated using Cox proportional-hazard models and consistency of treatment effect across subgroups was assessed using an interaction test. When patients were categorized by time from diagnosis, patients treated with selexipag earlier (time from diagnosis ≤6 months; N = 404) were younger and more likely to be in WHO FC II, treatment-naïve, and from Asia/Eastern Europe than those treated later (time from diagnosis > 6 months; N = 752). Selexipag reduced the risk of morbidity/mortality in patients treated earlier (HR: 0.45 (95% CI: 0.33–0.63)) and later (HR: 0.70 (95% CI: 0.54–0.91)), with a more pronounced treatment effect in those treated earlier (interaction p-value 0.0391). Consistent results were observed in subgroup analyses by background PAH therapy. Consistent with other studies suggesting that earlier initiation of treatment for PAH results in a more pronounced treatment effect, this analysis shows that outcome was better in patients who were treated with selexipag closer to the time of diagnosis. This pattern was observed in all background PAH therapy subgroups.

Maintenance dose reached and discontinuation of selexipag in patients treated for pulmonary arterial hypertension (PAH)

Harold I. Palevsky, Janis Pruett, Lin Xie, Adesuwa Ogbomo, Huiqi Wang, Rajesh Mallampati and Robert Dufour

University of Pennsylvania, Philadelphia, PA, USA; Actelion Pharmaceuticals US, Inc, South San Francisco, CA, USA; Statinmed, Ann Arbor, MI, USA

Selexipag is a prostacyclin receptor agonist approved by the US Food and Drug Administration (December 2015) for the treatment of PAH. As described in GRIPHON (pivotal long-term RCT for Selexipag), the package insert describes BID administration with a usual starting dose of 200 µg to individualized maximum tolerated dose up to 1600 µg BID. Selexipag is known to be efficacious at any individual maintenance dose in mono- and combination therapy. Currently, there is limited data available documenting real-world utilization of selexipag in the treatment of PAH. This observational, retrospective, baseline study used de-identified data (study period 1 January 2016 to 29 March 2019) from the specialty pharmacies dispensing selexipag to document duration of titration, maintenance dose, and persistence. The maintenance dose was determined as reached when the dose shipped was consistent (with BID dosing) for a 30-day supply. A total of 6709 patients had ≥1 selexipag prescriptions during the study period and 641 (9.6%) failed to meet the inclusion criteria. A total of 3931 patients (58.6%) reached a maintenance dose. Most (2058; 52.4%) attained a high dose (≥1200–1600 µg BID); the most frequent dose was 1600 µg BID (39.5%). The mean duration of titration was 14 weeks (97.7 days (64.2)) and 68.2% were persistent at six-months. However, 2137 (31.9%) patients did not reach a maintenance dose during the observation period and most (1517, 22.6%) discontinued. See Table 1 for other results. In conclusion, the maintenance dose for most patients was 1600 µg BID. However, many patients discontinued during titration and prior to reaching maintenance (22.6%). Recently, efforts are underway to educate patients and providers about the “dose adjustment” process to help patients reach “personalized dosing”. Further research to assess its impact on dosing, persistence, and discontinuation will be needed.

Table 1.

Summary of patient characteristics and results.

	Patients with a maintenance dose (n = 3931)	Patients excluded for not achieving a maintenance dose (n = 2137)
Mean age, years (SD)	57.9 (14.8)	61.3 (14.8)
Sex, female	73.0%	73.2%
Prior prostacyclin usage	13.3%	6.0%
Mean length of record, days (SD)	519.8 (323.7)	70.3 (128.7)
Up/down titration prior to maintenance dose	43.6%	NA
Time to maintenance dose, days (SD)	97.7 (64.2)	NA
Low dose (200–400 µg BID), n (%)	584 (14.9%)	NA
Medium dose (600–1000 µg BID), n (%)	1,289 (32.8%)	NA
High dose (1200–1600 µg BID), n (%)	2,058 (52.4%)	NA
Persistent at six months	68.2%	NA
Persistent at 12 months	48.4%	NA

NA, not applicable.

Insight into right ventricular defenses to pulmonary hypertension-associated pressure overload: potential role of PDZ protein EBP50

Maryam Sharifi-Sanjani, Mariah J. Berman, Patricia R. Marques, Adam L. Handen, Jeffrey J. Baust, Timothy N. Bachman, Andrea Sebastiani, Seyed M. Nouraie, Ana L. Mora, Stephen Chan and Imad Al Ghouleh

Division of Cardiology, Division of Pulmonary, Allergy and Critical Care Medicine, Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA, USA

Pulmonary hypertension (PH) is a progressive and fatal disorder characterized by high pulmonary arterial pressure that leads to right ventricular (RV) pressure overload and hence RV remodeling and eventual failure. While current PH drugs mainly target pulmonary vasculature, RV failure remains the leading cause of death for which no therapies exist. We recently showed that the scaffolding protein EBP50 (ERM-binding phosphoprotein 50) induces vascular cell hypertrophy. However, whether EBP50 plays a role in RV responses to pressure overload in vivo remains unknown. This study aims to evaluate whether genetic interruption of EBP50 exacerbates RV pressure (RVP) overload responses. Wild-type (WT) and EBP50 knockout (KO) mice were subjected to pulmonary artery (PA) banding (PAB), a procedure that constricts the PA, inducing pressure overload. Invasive hemodynamics were used for RV pressure–volume functional assessment. To glean mechanistic insight, we obtained gene expression profile (128 genes), utilizing a PCR array custom designed using in silico network analyses of curated PH-related and cardiomyopathy-related gene networks intersection. PAB induced a significant increase in systolic RVP and hypertrophy (Fulton index: RV/(left ventricle + septum) weights) in WT mice, which were further exacerbated in KO mice under PAB (RVP, mmHg: 43.98 ± 2.65 vs. 58.90 ± 2.36; Fulton index: 0.39 ± 0.02 vs. 0.5 ± 0.02, for WT PAB vs. KO PAB). RV gene expression arrays showed seven upregulated and 36 downregulated genes in WT PAB, some of which have not been previously connected to RV. Importantly, five of the seven upregulated genes were further enhanced in KO PAB, and 3 of the 36 downregulated genes were further attenuated in KO PAB. Taken together, our data demonstrate a novel role for EBP50 in modulating RV responses to RVP overload and offer mechanistic insight through identifying potential downstream targets. These findings have the potential to educate future development of RV-targeted therapies.

Familial pulmonary arterial hypertension by KDR heterozygous loss-of-function mutations

Florent Soubrier, Mélanie Eyries, Barbara Girerd, Nicolas Favrolt, Marianne Riou, Laurence Faivre, Grégoire Manaud, Frédéric Perros, Stefan Gräf, Nicholas W. Morrell, Marc Humbert and David Montani

Département de génétique, Hôpital Pitié-Salpêtrière, Assistance Publique-Hôpitaux de Paris, Paris, France; UMR_S1166-ICAN, Sorbonne Université, Paris, France; INSERM, Paris, France; Univ. Paris–Sud, Faculté de Médecine, Université Paris-Saclay, Le Kremlin Bicêtre, France; Assistance Publique Hôpitaux de Paris, Service de Pneumologie, Centre de Référence de l’Hypertension Pulmonaire, Hôpital Bicêtre, Le Kremlin Bicêtre, France; Service de Pneumologie et Soins Intensifs Respiratoires, Centre de référence constitutif des maladies pulmonaires rares de l’adulte, Centre de compétence de l’hypertension pulmonaire, CHU Dijon-Bourgogne, Dijon, France; Service de pneumologie, Nouvel hôpital civil, 1 place de l’hôpital, Strasbourg, France; Centre de génétique, FHU TRANSLAD, Institut GIMI et UMR INSERM 1231, CHU de Dijon et Université de Bourgogne, Dijon, France; UMR_S 999, Univ. Paris–Sud, Orsay, France; INSERM; Hôpital Marie Lannelongue, Le Plessis Robinson, France; Service de Chirurgie Thoracique, Hôpital Marie Lannelongue, Le Plessis Robinson, France; NIHR Bioresource – Rare Diseases, Departments of Medicine, Hematology, University of Cambridge School of Clinical Medicine, Addenbrooke’s and Royal Papworth Hospitals, Cambridge, UK

Beyond the major gene BMPR2, several new genes predisposing to PAH have been identified during the last decade. Recently, preliminary evidence of the involvement of the KDR gene was found in a large genetic association study using whole genome sequencing (Graf et al., PMID 29650961). We prospectively analysed the KDR gene by targeted panel sequencing in a series of 311 PAH patients referred to a clinical molecular laboratory for genetic diagnosis of PAH. Two index cases with severe PAH from two different families were found to carry a loss-of-function mutation in the KDR gene. These two index cases were clinically characterized by low diffusing capacity of the lungs for carbon monoxide (DLCOc) and interstitial lung disease (ILD) images at HRCT-scan. In one family, segregation analysis revealed that variant carriers are either presenting with declared PAH associated with low DLCOc, or have only a decreased DLCOc, whereas non-carrier’s relatives have normal DLCOc. In the second family, a single affected carrier was alive. His carrier mother is unaffected with normal DLCOc. We provide genetic evidence for considering KDR as a newly identified PAH causing gene by describing the segregation of KDR mutations with PAH in two families. In our study, KDR mutations are associated with a particular form of PAH characterized by low DLCOc and radiological evidence of ILD, opening the question whether KDR-linked PH should be considered as a heritable PAH form (§ 1.2 from the classification of PH) or as a developmental lung disorder PH (§ 3.5).

Single-cell RNA sequencing reveals the role of BMPR2/IDs signaling in CHD-PAH

Mingxia Du, Haibin Jiang, Hongxian Liu, Fang Zhou, Kezhou Qin, Hong Gu and Jun Yang

Department of Cell Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, Beijing, China; Department of Pediatric Cardiology, Beijing Anzhen Hospital, Capital Medical University, Beijing, China; Department of Physiology, Research Center of Molecular Medicine, Zhejiang University School of Medicine, Hangzhou, China

Recently, mutations in BMPR2 have been found in patients with congenital heart disease-associated PAH (CHD-PAH), especially in patients with postoperative PAH. Our study aimed to identify the molecular mechanism by which BMPR2 downstream signaling predisposes patients to CHD-PAH. Comparison of the expression of the Id genes, BMPR2 downstream effectors, between inducible pluripotent stem cell-cardiac mesoderm progenitors from healthy donors and CHD-PAHs with BMPR2 mutation, we showed a lower level of ID1 and ID3 total protein in CHD-PAHs, accompanied by a reduced cardiomyocytes (CMs) differentiation rate. Using human embryonic stem cell (hESC) lines with inducible overexpression (OE) of Id1 or Id3, we were able to show the reciprocal regulation between Id1 and Id3, and we unexpectedly found increased beating CMs formation during EC differentiation of the Id1 OE line. Double knockout (KO) of Id1 and Id3 in hESCs leads to decreased CMs differentiation. With single-cell RNA-seq analysis, we revealed deviated differentiation from cardiac progenitor cells (CPCs) and identified downregulated ubiquitin-specific protease (USP) expression in all clusters of KO lines compared with that in wild-type (WT) cells. Further analysis with the differentiated KO line revealed that CMs differentiation could be rescued by USP OE. In BMPR2 mutant CHD-PAH patients, we also found reduced USP expression. By using single-cell sequencing facilitated development-based analysis, we conclude BMPR2/ID1 signals through USP to drive CPC lineage determination during heart development in CHD-PAH with BMPR2 mutation. (This study was supported by National Key Research and Development Program of China-stem cell and translational research (2016YFA0102300); Nature Science Foundation of China (81870051, 81670054); and CAMS Innovation Fund for Medical Sciences (CIFMS) (2016-I2M-4-003).

Carbonic anhydrases 9 and 12 inhibition diminishes chronic hypoxia-induced pulmonary hypertension in mice

Aleksandar Petrovic, Djuro Kosanovic, Oleg Pak, Jan Ewert, Akylbek Sydykov, Henning Gall, Norbert Weissmann, Werner Seeger and Ralph T. Schermuly

Member of the German Center for Lung Research, Universities of Giessen and Marburg Lung Center, Giessen, Germany

Carbonic anhydrases (CAs) are family of zinc-containing metalloenzymes that reversibly catalyze carbon dioxide hydration to bicarbonate and protons. Over the last few years, CAs have been notably investigated in the cancer field, especially CA 9 and 12, as they are overexpressed in various types of tumors and often associated with disease progression and response to therapy. Pulmonary hypertension (PH) is a severe disease of various origins, characterized by several cancer-like features. Therefore, we hypothesize that CA 9 and 12 play an important role in the pathogenesis of PH. As our initial results revealed increased protein expression profile of both CA 9 and 12 in lung tissue samples derived from chronic hypoxia-induced PH in mice, we have investigated effect of CA 9 and 12 inhibition on disease development. In more detail, mice were randomized to treatment 21 days after normoxia or hypoxia exposure. Experimental groups included mice that received once-daily intraperitoneal injection of 100 mg/kg of S4 (potent small molecule CA 9 and 12 inhibitor) or administration of vehicle. Following two weeks of treatment, mice were subjected to the echocardiographic examination, hemodynamic measurement, right ventricular (RV) hypertrophy, and pulmonary vascular remodeling assessment. Our results demonstrated a significant decrease of RV systolic pressure in group of animals treated with S4, compared to their respective hypoxia controls. In addition, echocardiographic examination revealed significant effects of S4 treatment in various parameters, such as RV internal diameter and RV wall thickness. Finally, S4 treatment significantly reversed pulmonary vascular remodeling, as evident by significant decrease of percentage of fully muscularized vessels. In conclusion, our data demonstrated that pharmacological inhibition of CA 9 and 12 significantly attenuated chronic hypoxia-induced PH in mice. Finally, further investigation should reveal CA 9 and 12 potential as a therapeutic target in PH.

Association of right atrial reservoir phase with right ventricular function in pulmonary hypertension

Merle A. Wiegand, Manuel J. Richter, Henning Gall, Natascha Sommer, H.A. Ghofrani, Werner Seeger and Khodr Tello

Department of Internal Medicine, Justus-Liebig-University Giessen, Universities of Giessen and Marburg Lung Center, Member of the German Center for Lung Research, Giessen, Germany; Department of Pneumology, Kerckhoff Heart, Rheuma and Thoracic Center, Bad Nauheim, Germany; Department of Medicine, Imperial College London, London, UK

Awareness of right atrial (RA)–right ventricular (RV) axes in pulmonary hypertension (PH) is growing. Vascular remodeling with concomitant increased afterload eventually induces progressive RV maldapatation with impairment of RV function. RA speckle tracking has emerged as an important tool for the assessment of RA deformation and function. However, detailed association of RV function and RA phasic performance is currently unknown. Patients with PH from the RIGHT HEART I study (Unique identifier: NCT03403868) were included. RA reservoir function was prospectively assessed with echocardiographic speckle tracking analysis of peak longitudinal strain (PLS%). PLS% and B-type natriuretic peptide (BNP) were measured within 24 h of diagnostic right heart catheterization and conductance catheter with assessment of single-beat pressure-volume loops. Spearman rho correlation was used to determine the association of PLS% with RV function. In total, 51 patients (48 with pulmonary arterial hypertension and 3 with inoperable chronic thromboembolic PH) of whom the majority presented in WHO functional class III were analyzed. Elevated mean pulmonary pressure (rho = –0.336; p = 0.0016) and RV diastolic pressure (rho = –0.489; p < 0.001) were significantly associated with impaired RA PLS%. RA PLS% showed significant correlation with PVR (rho = –0.313; p = 0.025) and Tau (rho = –0.421; p = 0.002). In addition, BNP was associated with RA PLS% (rho = –0.486; p < 0.001). In conclusion, chronic pressure overload and increased afterload are associated with impaired RA reservoir function in PH. Strain analyses of RA reservoir function offer a novel tool to analysis impairment of the RA–RV axis.

Right ventricular dyssynchrony: association with load-independent right ventricular function in severe pulmonary arterial hypertension

Manuel Richter, Roberto Badagliacca, Jun Wan, Athiththan Yogeswaran, Hossein A. Ghofrani, Werner Seeger, Henning Gall, Robert Naeije, Merle A. Wiegand and Khodr Tello

Justus-Liebig-Universität Gießen, Gießen, Germany; Sapienza University of Rome, Rome, Italy; National Clinical Research Center for Respiratory Diseases, China-Japan Friendship Hospital, Beijing, China; Departments of Pneumology, Internal Medicine, University Hospital Brussels, Jette, Belgium

Coupling of the right ventricle to its load is considered to be one of the main determinants of symptomatology and outcome in pulmonary arterial hypertension (PAH). To maintain right ventricular (RV)–pulmonary arterial coupling, the right ventricle reacts with adaptation and eventually maladaptation as disease progresses. Among various maladaptive mechanism, RV dyssynchrony has been described as a relevant determinant of RV function, exercise capacity, and outcome in PAH. However, association of load-independent RV function with RV dyssynchrony remains unknown. We analyzed PAH patients from the Right Heart I study (ClinicalTrials.gov identifier: NCT03403868) with available 2D speckle-tracking echocardiography between January 2016 and March 2019. RV dyssynchrony was defined as the standard deviation of the times to peak systolic strain for the four mid-basal RV segments corrected to Bazett’s formula. Load-independent RV function was assessed due to acquisition of pressure–volume loops with single-beat measure of end-systolic elastance (Ees), arterial elastance (Ea), and the ratio of Ees/Ea (defines RV–pulmonary arterial coupling). Association between load-independent pressure–volume loop indices and RV dyssynchrony was evaluated with Spearman correlation coefficient and multivariate linear regression analysis. Thirty-three patients with PAH (idiopathic (n = 26; 79.8%)) were enrolled. The median RV dyssynchrony was 53 ms (interquartile range (IQR): 33–95 ms), Ees was 0.53 mmHg/ml (IQR: 0.30–0.76 mmHg/ml), Ea was 0.80 mmHg/ml (IQR: 0.49–1.10 mmHg/ml) and Ees/Ea was 0.71 (IQR: 0.38–1.04). Ea showed a significant correlation with RV dyssynchrony, albeit Ees or Ees/Ea not. In multivariate linear regression (including Ea, Ees, and Ees/Ea), Ea remained independently associated with RV dyssynchrony. In conclusion, Ea as a load-independent measure of afterload emerged as a determinate of RV dyssynchrony in advanced and severe PAH. The reduction of afterload might be the key strategy to improve RV dyssynchrony, which has to be explored in dedicated studies.

Pulmonary veno-occlusive disease, unexpected numbers in an extremely rare disease: a case series

Ojobumijo Agbaji, Brett Begley, Michael Ryan, Edward Murphy and Reda Girgis

Spectrum Health and Michigan State University, Grand Rapids, MI, USA

Pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis (PVOD/PCH) is a rare form of pulmonary hypertension without proven medical therapies. Risk factors include chemotherapy, organic solvents, smoking, connective tissue disease, and EIF2AK4 gene mutations in heritable cases. In this case series, we describe nine patients clinically diagnosed with PVOD, including two with pathologic confirmation. Each had at least two of the radiographic features on high-resolution computed tomography imaging and all had severe reduction (< 50%) in diffusion capacity for carbon monoxide (DLCO) and often profound hypoxemia. Median mean pulmonary artery pressure was 46 mmHg (range 31–49) and pulmonary vascular resistance 8.4 Wood Units (range: 3–15.5). Two subjects had underlying scleroderma, two had a known occupational exposure, and two had mild to moderate underlying emphysema. One subject had PAH diagnosed seven years previously and ultimately treated with triple oral PAH-targeted therapy. After biopsy confirmation of PVOD/PCH, a trial of interferon-alpha therapy has been initiated. The remaining eight were seen over approximately 24 months at our center serving a population of just over one million, which far exceeds the expected number of observed cases. Three patients died within six months of diagnosis, one is currently in home hospice, and one was lost to follow-up after denial for lung transplant consideration. One was successfully transplanted after 20 months during which she demonstrated a transient therapeutic response to low dose IV epoprostenol and one is currently listed for lung transplant. The last patient remains alive 25 months after diagnosis. She was treated with up-front triple combination therapy, including low-dose IV epoprostenol. This case series highlights the poor prognosis of the disease as well as the challenge and potential utility of PAH-targeted therapies. The relatively large number of cases seen over a short period of time raises the possibility of a common environmental factor in our geographic area.

Selexipag dosing and titration in the first 500 patients enrolled in SPHERE (SelexiPag: the users drug registry)

V. McLaughlin, N.H. Kim, A.R. Hemnes, K.B. Highland, K.M. Chin, H.W. Farber, C. Zhao, V Narayan, M. Shah and M.M. Chakinala

University of Michigan Medical Center, Ann Arbor, MI, USA; University of California, San Diego, La Jolla, CA; Vanderbilt University Medical Center, Nashville, TN, USA; Cleveland Clinic, Cleveland, OH, USA; Southwestern Medical Center, University of Texas, Dallas, TX, USA; Tufts, Boston, MA, USA; Actelion Pharmaceuticals US, Inc., South San Francisco, CA, USA; Washington University School of Medicine, St. Louis, MO, USA

SPHERE is an ongoing US-based, multicentre, prospective, registry collecting data on use of the oral selective IP prostacyclin receptor agonist selexipag in real-world settings. We report selexipag dosing and titration in the first 500 patients. SPHERE (NCT03278002) will enroll 800 patients newly initiated (≤60 days after starting selexipag) or previously initiated (> 60 days after starting selexipag) at enrolment with a documented titration regimen. Patients are followed for up to 18 months. The highest dose is the maximum dose reached during up-titration within six months since initiation. Selexipag “maintenance dose” is the first dose received for ≥14 days without interruption or change; “titration speed” is the highest dose divided by time (weeks) to reach it. Data cut-off for this analysis was 20 December 2018. Demographics and disease characteristics are summarized in Table 1. Median maintenance dose of selexipag was 1200 µg BID (IQR: 800–1600 µg BID); median time to reach it was 8.1 weeks (IQR: 5.3–11.0 weeks). Low (≤400 µg BID), medium (600–1000 µg BID), and high (≥1200 µg BID) maintenance doses were attained by 15.1%, 30.8%, and 49.5% of patients, respectively (and in 23.2%, 31.2%, and 36.2%, respectively, in GRIPHON). Median titration speed was 175 µg BID/week (IQR: 110.5–195.3 µg BID/week), equating to 200 µg BID increase every eight days, slightly slower than 200 µg BID every week (seven days) described in dosage and administration within FDA’s prescribing information. More patients discontinued due to adverse events in newly (29.0%) versus previously (14.1%) initiated groups, most commonly for worsening pulmonary hypertension (2.2%), headache (2.0%), myalgia (1.4%), and nausea (1.0%). Median maintenance selexipag dose in SPHERE was 1200 µg BID, median titration speed was 175 µg BID/week, and the vast majority of patients titrated slower than 200 µg BID/week. Safety profile was as observed in clinical trials.

Table 1.

Patient demographics and disease characteristics at selexipag initiation.

Characteristics	Patient (N = 500)
WHO group 1 (PAH) etiology, %	95.4
Idiopathic	49.6
Connective tissue disease	26.0
Other	24.4
Other WHO group etiology, %	4.6
WHO FC III at selexipag initiation, %	49.8
PH therapy with a PPA at selexipag initiation, %	19.2
Parenteral PPA	8.5%

PAH, pulmonary arterial hypertension; PH, pulmonary hypertension; PPA, prostacyclin pathway agent.

Advanced magnetic resonance imaging analytics for pulmonary artery hypertension

Yassine Abdledjebbar, Malik Bisserier, Michael Katz, Anthony Fargnoli, Sarah Gubara, Erik Kohlbrenner, Roger J. Hajjar and Lahouaria Hadri

Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York, NY, USA

Pulmonary arterial hypertension (PAH) is a fatal disease characterized by vascular remodeling which leads to the rise the pulmonary arterial pressure and ultimately right heart (RV) failure. Gene-based therapy is a potential new approach to treat PAH since it addresses root cause disease mechanisms of apoptosis and vascular smooth muscle proliferation in the lung vasculature. In this study, we evaluated SERCA2a gene therapy in severe pneumonectomy plus monocrotaline (MCT) (PNT-MCT) model of PAH with advanced MRI characterization of the left and RV. Following baseline MRI, contralateral PNT was performed in male Sprague Dawley rats. One week later, the animals received an injection of MCT. At three weeks, the severity of PAH disease was confirmed hemodynamically with MRI, then two groups of PNT-MCT rats received either intratracheal delivery of gene construct with adeno-associated virus/SERCA2a (AAV1/SERCA2a) or null vector. Hemodynamic parameters were determined by MRI and RV catheterization at six weeks post baseline. Four weeks after gene delivery, the global RV function was improved in AAV1.SERCA2a-treated animals with an increase of stroke volume and ejection fraction compared to saline group (275 ± 22 ± vs. 192 ± 22 µL and 56 ± 3 vs. 44 ± 3%, p < 0.05), while RV end-systolic volume was decreased (199 ± 16 vs. 283 ± 24 µL, p < 0.05). Hemodynamic parameters including mean pulmonary pressure were improved in AAV1.SERCA2a group (26 ± 3 mmHg vs. 61 ± 6 mmHg and 21 ± 3 mmHg vs. 41 ± 3 mmHg respectively, p < 0.01) compared to control. At six weeks, the SERCA2a gene therapy group measured 4.4 ± 0.1 mm vs. 5.3 ± 0.1 mm in controls, p < 0.01 as compared to a baseline of 4 mm confirming the reversal of dilation. The right ventricular lengths measured from the tricuspid valve to apex were affected: Control at six weeks 12.0 ± 0.2 mm vs. SERCA2a at six weeks 15.6 ± 1.1 mm as compared with baseline 15.1 ± 0.3 mm. These data taken together demonstrate the value in tracking RV length and width dimensions which positively correlate with ejection fraction. Additionally, TAPSE long axis plane calculations confirmed SERCA2a rescued function with a displacement of 4.1 ± 0.6 mm vs. six weeks control of 2.7 ± 0.4 mm against a baseline reference 5.2 ± 0.2 mm, p < 0.05. Histologically, the animals after gene therapy showed a significant regression of plexiform lesions from grade 4 to grade 1–2. In conclusion, intratracheal administration of AAV1/SERCA2a gene can reverse the severe PAH phenotype and may be considered as a potential treatment. MRI is a powerful tool to quantify the degree of PAH rescue over time and represents a major achievement to improve the execution of translational studies.

Case study of long-term safety, tolerability, and hemodynamic response of Pb1046, a sustained-release analogue for vasoactive intestinal peptide (VIP), in an adult subject with pulmonary arterial hypertension (PAH)

Raymond L. Benza, Sumita Paul, Murali Chakinala, Priscilla Correa, John Lee and James White

The Ohio State University Wexner Medical Center, Columbus, OH, USA; University of Rochester, Rochester, NY, USA

PB1046 is an investigational neuropeptide, vasoactive intestinal peptide (VIP), genetically fused to an elastin-like biopolymer, for treatment of PAH, with biologic effects mediated by two receptors, VPAC1 and VPAC2, belonging to the family B of G protein-coupled receptors. VPAC receptors are in the pulmonary and systemic circulation. The safety and accuracy of the CardioMEMS™ HF System, which monitors pulmonary artery (PA) pressure from a sensor implanted into the PA, have been previously documented, along with correlations with Swan-Ganz measurements and echocardiography. The multi-dose safety, PK, and VIP-based pharmacodynamic effects of PB1046 were evaluated in an open-label, multi-dose Phase 1 pilot study in PAH patients who have a permanently implanted CardioMEMS™. PB1046 was administered weekly subcutaneously for eight weeks (extended due to subjective improvements) at dose levels previously tested and shown to be safe. Three patients completed study with no related serious adverse events/symptomatic hypotension/syncope. PB1046 appears to be well-tolerated with only mild injection site erythema. The available PK profile data confirmed the dose-related but less than dose-proportional increase in study drug exposure profile observed in previous studies. CardioMEMS™ hemodynamic monitoring demonstrated reductions in mean PA pressure and total pulmonary resistance and increases in stroke volume and cardiac output without an increase in heart rate with PB1046. Long-term PB1046 therapy (over 18 months) in one subject demonstrated clinically meaningful improvements in all the hemodynamic parameters assessed, which were sustained for three months afterwards, suggesting a possible disease-modifying effect of PB1046 (Fig. 1) and will be presented. The preliminary data for PB1046 support continued evaluation as a potential novel therapy for PAH patients that is safe and well-tolerated. Improvements in hemodynamic parameters associated with PB1046 support the ongoing randomized, double-blind, parallel group Phase II PAH study assessing 16 weeks of PB1046 treatment for NYHA/WHO functional classes II and III PAH patients.

Fig. 1.

Effect of PB1046 on hemodynamic parameters.

Comparison of expert-derived versus machine-generated survival model for pulmonary arterial hypertension survival

Manreet K. Kanwar and Raymond L. Benza

Department of Medicine, Allegheny General Hospital, Pittsburgh, PA, USA; Department of Medicine, The Ohio State University Wexner Medical Center, Columbus, OH, USA

While risk assessment scores for stratifying pulmonary arterial hypertension (PAH) patients have been around for many decades, clinicians frequently rely on gestalt for assessing risk. The goal of this work was to benefit from the deep clinical knowledge of clinical experts to derive a predictive model for PAH. We then compared the performance of this hybrid model to one derived exclusively by machine learning. Eleven clinicians were presented with a diagram containing 14 predictor variables and invited to draw arcs (lines) indicating causal connections among them, and with the outcome of survival at 12 months. These variables were the same as found in the REVEAL 2.0 calculator, although the clinicians were not required to use every variable. After creating networks that represented their understanding of causality for mortality risk, each network was programmed as a Bayesian network, using a modeling user interface (GeNIe). A combined expert model was also developed, using arcs that were common to at least two individual experts. Independently, the parameters were learned using the REVEAL registry data from patients at 12 months post enrollment. Each model was evaluated using 10-fold cross validation and the areas under the receiver-operator characteristic curve (AUCs) were compared. Seventeen expert models were created (some clinicians made multiple versions) and their performance ranged from an AUC 0.66–0.73 (Table 1). The combined expert model performed with an AUC of 0.72. The model learned from data using a tree-augmented naïve Bayesian network performed with an AUC of 0.86. In conclusion, experts do not agree on what parameters are most related to survival outcomes. Networks built by the experts varied in the quantity of variables and the relationships among them. A combination of expert models gave a reasonably accurate model, but its performance was inferior to the machine-learned model. There is a need for inclusion of mathematically derived risk assessments to complement the clinicians’ assessment of patients.

AUC	No. of arcs in network	Top 3 predictive variables
0.66	22	Estimated glomerular filtration rate (eGFR), Systolic BP, age
0.69	28	WHO category, all cause hospitalization, pericardial effusion
0.70	28	WHO category, all cause hospitalization, pericardial effusion
0.70	34	WHO category, all cause hospitalization, pericardial effusion
0.70	40	WHO category, all cause hospitalization, pericardial effusion
0.70	41	WHO category, all cause hospitalization, pericardial effusion
0.71	18	Pericardial effusion, all cause hospitalization, 6MWD
0.71	26	All cause hospitalization, 6MWD, pericardial effusion
0.72	27	WHO category, all cause hospitalization, pericardial effusion
0.72	27	WHO category, all cause hospitalization, pericardial effusion
0.72	25	WHO category, pericardial effusion, all cause hospitalization
0.73	32	All cause hospitalization, BNP, 6WMD
0.73	30	All cause hospitalization, WHO category, 6MWD
0.73	14	All cause hospitalization, pericardial effusion, 6MWD
Combined network:
0.72	43	All cause hospitalization, 6WMD, B-type natriuretic peptide (BNP)

6MWD, six-minute walking distance.

Comparisons of the efficacy of mono or combination macitentan treatments in idiopathic and congenital heart disease-associated pulmonary arterial hypertension

Cihangir Kaymaz, Seda Tanyeri, Ozgur Y. Akbal, Berhan Keskin, Aykun Hakgor, Ali Karagoz, Hacer C. Tokgoz, Zubeyde Bayram, Seyhmus Kulahcioglu, Cem Dogan, Emrah Erdogan, Rezzan D. Acar, Ibrahim H. Tanboga and Nihal Ozdemir

Kartal Kosuyolu Heart Education and Research Hospital, University of Health Sciences, Istanbul, Turkey; Department of Biostatistics, Medical School, Ataturk University, Erzurum, Turkey; Department of Cardiology, Hisar Intercontinental Hospital, Nisantası University, Istanbul, Turkey

In this study, we aimed assess the effects of macitentan treatment (MaciT) on clinical, echocardiographic, neurohumoral measures in treatment-naive (TN) and non-naive (NN) patients with idiopathic pulmonary arterial hypertension (IPAH) and congenital heart disease-associated-PAH (CHD-PAH). Moreover, relation between longitudinal changes of these measures and mortality was analysed. We evaluated 175 patients (age 45.1 ± 17.9, female 74%) with IPAH (74, 42.2%) or CHD-PAH (87, 49.7%) who were diagnosed according to current guidelines and followed-up prospectively after initiation of MaciT as mono or part of dual or triple combination therapies between 2013 and 2018. Functional-class (FC), six-minute walking distance (6MWD), pericardial effusion (PE), right atrial area (RAA), tricuspid annular plane systolic excursion (TAPSE), systolic-motion (St) and brain natriuretic peptide (BNP) measures were evaluated at baseline and periodical control examinations with six months apart. Longitudinal changes in these measures were evaluated with generalized linear mixed model, and relationship between these changes and mortality were analysed with Bayesian joint modelling. Age and female gender were 45.6 ± 17.6 and 58 (78.3%) in IPAH, and 41.4 ± 16.8 and 60 (68.9%) in CHD-PAH patients, respectively. Median follow-up period was 16 months. TN and NN patients were 73 and 102, and MaciT was noted as monotherapy in 25 (14.3%) patients, and part of dual or triple combinations in 100 (57.2%) and 50 (28.5%) patients, respectively. The FC, 6MWD and TAPSE, but not PE, RAA and BNP showed statistically significant improvements after MaciT. Bayesian joint modelling revealed that age (hazard ratio (HR): 1.04; 95% confidence interval (CI): 1.00–1.07) and male gender (HR: 5.12; 95% CI: 1.63–16.1) IPAH versus CHD-PAH (HR: 2.11; 95% CI: 0.71–6.32), longitudinal changes in FC (HR: 1.09; 95% CI: 0.86–1.40), 6MWD (HR: 0.99; 95%CI: 0.99–1.00), TAPSE (HR: 1.00; 95%CI: 0.97–1.03) after MaciT were not associated with mortality. Moreover, TN or NN status, or mono or combination, were not associated with effects of MaciT. Side-effect requiring drug cessation was not documented. MaciT seemed to provide comparable benefits for clinical, echocardiographic and neurohumoral outcome measures in IPAH and CHD-PAH, regardless of the TN or NN status or mono or combination therapies. However, age and male gender, but not these improvements, were associated with mortality in patients under MaciT.

Targeting peptidyl-prolyl isomerase 1 in experimental pulmonary arterial hypertension

Nabham Rai, Akylbek Sydykhov, Baktybek Kojonazarov, Jochen Wilhelm, Hossein A. Ghofrani, Norbert Weissmann, Werner Seeger, Tatyana Novoyatleva and Ralph T. Schermuly

Universities of Giessen and Marburg Lung Center, Excellence Cluster Cardio Pulmonary Institute, Member of the German Center for Lung Research, Justus-Liebig-University Giessen, Giessen, Germany; Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany

Pulmonary arterial hypertension (PAH) is a progressive disease of various origins that is associated with a poor prognosis and results in right heart dysfunction. An increase in vascular cell proliferation and their resistance to apoptosis are prominent features of pulmonary vascular remodeling in PAH. Proline (Pro)-directed Ser/Thr phosphorylation (pSer/Thr-Pro) is a common signaling mechanism in cells. Peptidylprolyl cis/trans isomerase, NIMA interacting 1 (PIN1), a highly conserved enzyme which binds to and catalyzes the isomerization of specific phosphorylated Ser/Thr-Pro motifs, acting as a molecular switch in coordinated cellular processes. Our aim is to identify the impact of PIN1 on the emergence of PAH. Immunofluorescence, RT-qPCR, and western blot analyses were performed for expression studies. Inhibition of PIN1 in human pulmonary vascular cells has been achieved by siRNA targeting knock-down or small molecule inhibitor (Juglone) approaches. Functional analysis involved proliferation and apoptosis assays. Screening of activation of transcription factors was performed by luminescence assay. Sugen5416/hypoxia rat and chronic hypoxia mouse experimental PAH models were entailed for in vivo studies. Upon investigation, PIN1 was found to be up-regulated in lungs and pulmonary artery smooth muscle cells of patients with idiopathic PAH, as well as in studied experimental PH models. In vitro PIN1 deletion by either Juglone treatment or siRNA-knock-down resulted in a decrease in proliferative response and inhibition of resistance to apoptosis of human pulmonary vascular cells. Stimulation with pro-proliferative growth factors induced PIN1 expression, while its deletion reduced the activity of key PAH-related transcription factors (HIF, STATs, etc). In vivo studies showed that Juglone lowered pulmonary vascular resistance, improved RV function, and reduced pulmonary vascular remodeling in both Sugen5416/hypoxia and chronic hypoxia models. These results indicate that targeting of PIN1 may be potentially clinically beneficial in the treating of PAH and right heart disease secondary to PAH.

Regorafenib, a multikinase inhibitor therapy for pulmonary arterial hypertension

Swathi Veeroju, Tatyana Novoyatleva, Baktybek Kojonazarov, Astrid Weiss, Hossein A. Ghofrani, Friedrich Grimminger, Norbert Weissmann, Werner Seeger and Ralph T. Schermuly

Department of Internal Medicine, Universities of Giessen and Marburg Lung Center (UGMLC), Cardio-Pulmonary System (CPI), Member of the German Center for Lung Research (DZL), Justus-Liebig-University, Giessen, Germany; Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany

Pulmonary arterial hypertension (PAH) is characterized by elevated mean arterial pressure followed by right ventricular failure and death. Previous studies have shown that targeting receptor tyrosine kinases (RTKs) is beneficial in the treatment of pulmonary hypertension. Regorafenib (BAY 73-4506) is a kinase inhibitor that targets PDGFR, RET, C-KIT, FGFR, RAF-1, VEGFR 1–3, and p38 MAP kinases. Regorafenib has been clinically approved for the treatment of both metastatic colorectal cancer and gastrointestinal stromal tumors in Imatinib and Sunitinib non-responders. As PDGFRβ and p38 MAPK inhibition were known to reverse PAH phenotype and Regorafenib has demonstrated PDGFRβ and P38 MAPK inhibitory effects in numerous studies, we hypothesized the potential of Regorafenib as a therapeutic strategy for PAH. In vitro functional parameters were studied by proliferation and migration assays in pulmonary arterial smooth muscle cells (hPASMCs) of non-PAH controls and patients with idiopathic PAH (IPAH). Proliferation and migration, induced by platelet-derived growth factor (PDGF-BB) were determined by 5-bromo-2’-deoxyuridine (BrdU) incorporation assay and Transwell Chamber assays, respectively. Effect on RTK activity in hPASMCs was analyzed by Western Blot analysis. In vivo studies include chronic hypoxic mice (HOX)- and monocrotaline (MCT)-induced rat PH models. Functional parameters were analyzed by hemodynamic and echocardiographic measurements. Vascular remodeling was studied by degree of muscularization. Regorafenib demonstrated strong anti-proliferative effects on PDGF-BB-induced proliferation in both non-PAH and IPAH hPASMCs. Anti-migratory effects were observed in non-PAH hPASMCs in a dose-dependent manner. Regorafenib has shown strong inhibition of RTKs like p38 MAPK and PDGFRβ in hPASMCs. Although reduced pulmonary vascular remodeling was observed only in HOX model, functional parameters demonstrated improved cardiac function with reduced pulmonary vascular resistance upon Regorafenib treatment in both in vivo models. Our data suggest that treatment with Regorafenib can be a novel therapeutic approach for the treatment of PAH.

Long-term effect of exposure to e-cigarettes vapour in mice

Oleg Pak, Elsa T. Roxlau, Alexandra Pichl, Claudia G. Castro, Mariola Bednorz, Katharina Schäfer, Stefan Hadzic, Simone Kraut, Jochen Wilhelm, Ralph T. Schermuly, Hossein A. Ghofrani, Werner Seeger, Friedrich Grimminger, Natascha Sommer and Norbert Weissmann

Cardio-Pulmonary Institute, University of Giessen and Marburg Lung Center, Member of the German Center for Lung Research, Giessen, Germany

Electronic cigarettes (e-cigarettes) are gaining popularity as an alternative to smoking, despite many unsolved questions regarding their safety. Compared to the smoke from conventional cigarettes, inhaled e-cigarette vapour (ECV) contains less toxic substances. However, limited data are available in particular on long-term effects of ECV on the lung. Effects of exposure to ECV extract (ECVE) with or without nicotine (18 mg/ml) were studied in isolated murine pulmonary arterial smooth muscle cells (PASMC) and alveolar epithelial type II (ATII) cells and compared to effects of conventional cigarette smoke extract. Lung emphysema and pulmonary hypertension (PH) were investigated in mice after exposure for eight months (6 h/day, 5 days/week) to ECV with (18 mg/ml) or without nicotine. Result shows that ECVE with and without nicotine decreased cellular viability of PASMC and ATII cells and proliferation of PASMC. Microarray analysis showed that genes related to the cell cycle, DNA replication and spliceosome were upregulated, while genes of lysosomal and metabolic pathways were downregulated in PASMC exposed to ECVE with or without nicotine. Exposure of wild-type mice (C57BL/6J) to ECV with or without nicotine did not affect parameters defining emphysema or PH compared to room air-exposed mice. However, long-term exposure to ECV altered the number of neutrophils, lymphocytes and macrophages in the bronchoalveolar lavage fluid. In conclusion, exposure to ECVE alters several signalling pathways in murine pulmonary cells independent of the nicotine content. Long-term exposure of mice to ECV does not result in functional and structural pulmonary alterations, but induces an inflammatory response in the concentration applied. Further experiments are necessary to understand the effect of e-cigarette exposure on human health, in particular different smoking regimes should be investigated including higher dose ECV exposure as may occur during e-vapour inhalation in humans.

Of mice and rats – profound species differences in the capacity for lung microvascular repair after widespread lung endothelial injury and apoptosis

Rafael S. Godoy, Mohammed Taha, Yupu Deng, Katelynn Rowe, Nicholas Cober and Duncan J. Stewart

Sinclair Center for Regenerative Medicine, Ottawa Hospital Research Institute, Ottawa, Canada; Faculty of Medicine, University of Ottawa, Ottawa, Canada

We studied mechanisms of lung microvasculature repair in two models of endothelial cell (EC) injury: a murine transgenic model for the diphtheria toxin (DT) mediated ablation of ECs and a rat model treated with the VEGFR2 inhibitor, SU5416 (SU). After a single dose of 10 ng DT, transgenic mice expressing the human DT receptor targeted to ECs showed an increase in activated caspase 3 and TUNEL labeling in lung ECs at 48 and 72 h post treatment compared to saline controls, resulting in a 78 ± 10% (p < 0.001) decrease CD144⁺ lung ECs by flow cytometry. This was associated with a 35 ± 8% decrease in total lung arterial volume assessed by micro-CT, mainly in the microcirculation (vessels between 50–150 µm in diameter). Right ventricular systolic pressure (RVSP) was increased at 72 h in DT vs. saline-treated diphtheria toxin receptor (DTR) mice (RVSP: 34 ± 1 vs. 24 ± 0.35 mmHg, respectively, p < 0.0001, n = 19), whereas right ventricular hypertrophy (n =1 5) was only seen at one week (0.27 ± 0.006 vs. 0.24 ± 0.004; respectively; p = 0.001). Remarkably, there was a full recovery in EC number and microvascular volume by one week after DT treatment, with normalization of RVSP and lung morphology, consistent with efficient microvascular repair. Rats treated with a single injection SU5416 (20 mg/kg) showed a similar increase in RVSP at one week (39 ± 5 mmHg vs. 28 ± 4 mmHg, SU and control, respectively, n = 9, p = 0.001) and a ∼40% reduction in total lung arterial volume by micro-CT (6.1 e¹¹ ± 9.1 e¹⁰ vs. 3.8e¹¹ ± 2.5e¹⁰ µm³, p = 0.001). However, in contrast to mice, rats did not show any significant recovery at three or five weeks following SU administration for RVSP (39 ± 2 mmHg and 40 ± 2 mmHg, respectively) or microvascular loss (4.5e¹¹ ± 4e¹⁰ and 4.6e¹¹ ± 4e¹⁰ µm³, NS vs. one week 3.8e¹¹ ± 2.5e¹⁰ µm³). In conclusion, the capacity for lung microvasculature repair was markedly greater in mice vs. rats, possibly contributing to differences in susceptibility to PAH induced by EC injury.

Mitochondrial alternative oxidase inhibits acute, but not chronic hypoxic signaling of the pulmonary vasculature

N. Sommer, N. Alebrahimdehkordi, O. Pak, F. Knoepp, S. Scheibe, A. Sydykov, A. Saraji, Simone Kraut, Matthias Hecker, K. Quanz, Joel Wahl, W. Seeger, H.A. Ghofrani, R.T. Schermuly, F. Grimminger, Kerstin Ramser, T. Braun, H.T. Jacobs, N. Weissmann and M. Szibor

Excellence Cluster Cardio-Pulmonary Institute, University of Giessen and Marburg Lung Center, Member of the German Center for Lung Research, Justus-Liebig-University Giessen, Giessen, Germany; Department of Engineering Sciences and Mathematics, Luleå University of Technology, Luleå, Sweden; Faculty of Medicine and Health Technology, BioMediTech & Tampere University Hospital, Tampere University, Tampere, Finland; Department I, Cardiac Development and Remodelling, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany

Acute alveolar hypoxia results in hypoxic pulmonary vasoconstriction (HPV), while chronic hypoxic exposure leads to pulmonary hypertension (PH). We have recently shown that mitochondrial reactive oxygen species (ROS) release triggers HPV, but not PH, via a mechanism depending on mitochondrial complex IV subunit composition. We now aimed to investigate if inhibition of mitochondrial respiration and subsequent accumulation of electrons distal of complex IV may act as trigger mechanism for acute hypoxia-induced ROS release and HPV. We used mice expressing an alternative oxidase (AOX) which can pass electrons directly from complex I or II to oxygen, when either complex III or IV is inhibited thereby avoiding electron accumulation distal from complex IV. HPV was investigated in isolated lungs, and PH in mice exposed to four weeks of hypoxia. Superoxide levels of isolated pulmonary arterial smooth muscle cells (PASMC) were measured by electron spin resonance spectroscopy, cellular redox state by RAMAN spectroscopy, and cellular membrane potential (as cellular HPV equivalent) by patch clamp. Bypassing complex III/IV and maintaining electron flow through complex I/II by AOX expression, inhibited acute hypoxia-induced increase of NADH/NAD and superoxide levels, cellular membrane depolarization of PASMC and HPV in isolated lungs, whilst the response to a hypoxia-independent vasoconstrictive stimuli was preserved. In contrast, chronic hypoxia-induced PH in mice, and stabilization of the hypoxia-inducible factor 1α in PASMC were not affected by AOX expression. These results confirm our previous observation that mitochondrial ROS release is essential for acute, but not chronic hypoxic signaling in the pulmonary vasculature. Inhibition of mitochondrial respiration resulting in electron accumulation at complex I/III may act as initial trigger for hypoxia-induced superoxide release and HPV.

The effects of increasing doses of pulsed iNO on pulmonary arterial compliance (PAC) measured by right heart catheterization (RHC) in patients with pulmonary hypertension (PH) associated with pulmonary fibrosis (PF)

R. Alvarez, R. Dudenhofer, K. Ahmad, M. Glassberg, L. Lancaster, G. Raghu, J. Stewart, P. Fernandes, H. Gillies, P. Shah, R. Baughman and S.D. Nathan

University of Miami, Coral Gables, FL, USA; Vanderbilt University Medical Center, Nashville, TN, USA; INOVA Heart and Vascular Institute, Annandale, VA, USA; University of Arizona, Phoenix, AZ, USA; University of Washington Medical Center, Seattle, WA, USA; Temple University Hospital, Philadelphia, PA, USA; Bellerophon Therapeutics, Warren, NJ, USA; University of Cincinnati, Cincinnati, OH, USA

Pulmonary fibrosis (PF) is made up of a wide variety of fibrotic lung diseases. Pulmonary hypertension (PH) frequently complicates pulmonary fibrosis (PH-PF) and is associated with worsening clinical outcomes. There are currently no approved therapies to treat PH-PF. PAC describes the pulsatile afterload that accounts for approximately 25% of the total RV afterload, and a reduction in pulmonary arterial compliance (PAC) may initiate and/or exacerbate distal pulmonary vasculopathy and RV–PA uncoupling. PAC has been shown to be a strong predictor of outcomes in PAH. None of the currently available PAH pulmonary vasodilator therapies cause consistent and meaningful improvements in PAC. There is limited data on changes in PAC in PH-PF patients. The aim of this study was to determine if increasing doses of inhaled nitric oxide (iNO) can improve PAC in patients with PH-PF. PAC was evaluated in PF-PH patients undergoing RHC. PAC was derived as Stroke Volume/(Systolic PAP – Diastolic PAP). Increasing doses of pulsed iNO, 30, 45, and 75 mcg/kg-IBW/h, were administered over a 10-min period, followed by a 10-min washout. On completion of the RHC, subjects were offered the opportunity to go on to chronic iNO therapy in an extension study. Preliminary results are presented for the change from baseline in PAC for the first four subjects enrolled in the study (total N = 8). Four subjects with PF-PH with a mPAP 34 mmHg, cardiac index 1.7 L/min/m², PVR 584 dyne*s/cm⁵, PCWP 10.8 mmHg, and PAC 2.2 ml/mmHg at baseline demonstrated a reduction in PAC with increasing doses of pulsed iNO (Fig. 1). PAC improved after acute dosing with pulsed iNO. The iNO was safe and well tolerated. Subjects will be followed in an extension study where the acute change in PAC at baseline will be correlated with long-term change in symptoms.

Fig. 1.

The change in PAC (m./mmHg) by dose.

Use of pulmonary arterial hypertension (PAH)-specific pharmacotherapies among 111,356 veterans with pulmonary hypertension

Thomas M. Cascino, David W. Schopfer, Craig S. Meyer, Ning Zhang, Vallerie V. McLaughlin and Mary A. Whooley

Division of Cardiovascular Medicine, University of Michigan, Ann Arbor, MI, USA; San Francisco Veterans Affairs Medical Center, San Francisco, CA, USA; Department of Medicine, University of California, San Francisco, CA, USA

Guideline consensus regarding the use of PAH-specific therapies for the treatment of pulmonary hypertension (PH) secondary to left-heart disease (WHO group 2) or chronic lung disease (WHO group 3) has moved from potential benefits, to limited use by expert centers, to recommendations against use. We sought to understand the trends in the real-world use of PAH-specific therapies among a cohort of patients with PH. We identified patients with incident PH from the Veterans Affairs healthcare system from 2010 to 2016 using a validated claims-based algorithm. Patients were classified by etiology of PH: WHO group 1 (PAH), groups 2–4 (CTEPH), or unknown. Patients could belong to more than one group, with the exception of group 1. The use in PAH-specific pharmacotherapies in the first year after diagnosis and trends in the rates of use of pharmacotherapies from 2010 to 2016 were determined. Results showed that among 111,356 incident cases of PH, 7799 (7.0%) had group 1 PH, 71,615 (64.3%) had group 2 PH, 43,115 (38.7%) had group 3 PH, 4084 (3.7%) had group 4, and 16,883 (15.2%) were unknown. A PAH-specific medication was used in 1358 (17.4%) of group 1, 7616 (10.6%) of group 2, 5948 (13.8%) of group 3, 499 (12.2%) of group 4, and 2395 (14.2%) of the unknown group. From 2010 to 2016, there was no increase in pharmacotherapy use (P = 0.86) among group 1 patients while there was an increase in use among groups 2 and 3 patients (P < 0.001). We found increasing use of PAH-specific pharmacotherapies among patients with groups 2 and 3 PH despite guidelines evolving to recommend against use over a similar time period. A greater understanding of the reasons for potentially inappropriate use would allow targeted interventions aimed at reducing misuse.

Wipi1 is a conserved mediator of right ventricular failure and potential target for novel therapeutic development

Christos Tzimas, Christoph Rau, Petra E. Buergisser, Jr Gaston Jean-Louis, Katherine Lee, Jeffrey Chukwuneke, Wen Dun, Yibin Wang and Emily J. Tsai

Division of Cardiology, Columbia University Vagelos College of Physicians and Surgeons, New York, NY, USA; Division of Molecular Medicine, Department of Anesthesiology and Perioperative Medicine, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA

Right ventricular dysfunction (RVD) is highly prevalent across cardiopulmonary diseases and independently predicts worse outcomes, including increased mortality rate, in both heart failure (HF) and pulmonary hypertension (PH). The prognostic significance of RVD holds true in HF and PH, regardless of left ventricular ejection fraction and pulmonary artery systolic pressure, respectively. Moreover, progression toward RVD and right ventricular failure (RVF) can occur in spite of optimal medical treatment of HF or PH, highlighting the limitations of our current understanding of RVF molecular pathophysiology. The aim of this study was to identify molecular mechanisms that may distinctly underlie RVF, we investigated the cardiac ventricular transcriptome of advanced HF patients, with and without hemodynamically significant RVF. Using weighted gene co-expression network analysis and module-phenotype correlations, we identified an RVF-specific gene module, for which WIPI1 served as a hub and HSPB6 and MAP4 as drivers. We then confirmed the ventricular specificity of RVF-associated upregulation of Wipi1, Hspb6, and Map4 in adult murine models of pressure overload induced RV- versus LV failure. We uncovered a shift toward non-canonical autophagy in the failing RV that correlated with RV-specific Wipi1 upregulation. In vitro siRNA silencing of Wipi1 in neonatal rat ventricular myocytes limited non-canonical autophagy and blunted aldosterone-induced mitochondrial superoxide levels. Our findings suggest that Wipi1 regulates mitochondrial oxidative signaling and non-canonical autophagy in cardiac myocytes. Together with our human transcriptomic analysis and corroborating studies in an RVF mouse model, these data render Wipi1 a potential myocardial target for RVF therapy.

The right ventricle ejection fraction (RVEF) for heart gammagraphy for chronic obstructive pulmonary disease (COPD), in a third level hospital in the altitude of Quito-Ecuador

Rodrigo P. Hoyos, Miriam T. Muñoz and Elena M. Ortega

Pneumology Technical Unit, Carlos Andrade Marín Specialty Hospital, Quito, Ecuador; Cardiology Technical Unit, Carlos Andrade Marín Specialty Hospital, Quito, Ecuador

Quito has 2,644,145 people. It is located at 2840 m above sea level. The group of patients with COPD is a group of high demand in the Health Systems of the country; factors such as altitude level, right ventricular ejection fraction (RVEF), and Hematocrit, directly influence. The RVEF by radionuclide ventriculography or radionuclide angiography (MUGA) was used due to the high demand for echocardiograms in our hospital, despite not being recommended in the international consensus. Retrospective observational study, case series; with a population and known sample of 17 patients, inclusion criteria: patients with a diagnosis of COPD who attended the pneumonology external consultation of the Carlos Andrade Marín Specialties Hospital (HCAM), in 2016, in the city of Quito, Ecuador in 2016. The data were obtained from the electronic medical history of the AS400 system, over 18 years old, both genders. Among the exclusion criteria were healthy patients and loss of data. The data were analyzed with the help of the Excel system, and SPPS.23.0, validating the RVEF, left ventricular ejection fraction, correlated with other variables: age, gender, hemodynamic values, and residence in meters above the level of the sea (MASL) in each group of COPD according to its GOLD. Results showed that the age range of patients who attended the consultation with a diagnosis of COPD was 51–88 years. Male gender with 70.6% (12–17) and female 29.4% (5–17), predominates COPD GOLD 2 (Fig. 1a and b). The majority of patients live in Quito between 1001-3000 MASL. The LVEF was calculated in the ranges 21–80, and RVEF with a range between 24 and 66 mmHg and predominance of the NYHA II functional class (Figs 2 to 5). The mean age in the case series is 51 years. The majority of patients are male and with COPD GOLD 2, with NYHA 2 functional class, live between 1001 and 3000 m.a.s.l. There were no cases in NYHA 4 functional capacity or with COPD GOLD 4, they probably do not move from their homes. The LVEF is not modified, it is maintained at normal values (above 50–55%). The forced expiratory volume in one second (FEVD) decreases progressively as the GOLD and altitude increase; however, in the present case series, there are normal values (above 40–45%). The etiology of PH in COPD is multifactorial. This series of cases is the beginning of future research to promote adequate education in chronic diseases of altitude and high altitude. The Multiple Gated Equilibrium Cardiac Blood Pool Scintigraphy technique (MUGA) for measuring RVEF in patients with COPD increases suspicion of right heart size increase and then to confirm with echocardiogram, cardiac magnetic resonance, and right heart catheterization.

Fig. 1.

(a) Age. Source: Data base of Investigation. (b) Gender. Source: Data base of Investigation.

Fig. 2.

Data of meters above sea level. Source: Data base of Investigation.

Fig. 3.

Data according to RVEF. Source: Data base of Investigation. Produced by authors.

Fig. 4.

Data according to LVEF. Source: Data base of Investigation. Produced by authors.

Fig. 5.

GOLD 2 hemodynamic parameters. Source: Data base of Investigation. Produced by authors.

Severe pulmonary hypertension in patient with D-transposition of large arteries and intact ventricular septum: patience and multimodal treatment, the best counselors

Luis E. Ponce, Walter Mosquera, Jaiber Gutierrez, Karen Molina and Carlos Gonzalez

Universidad ICESI, Cali, Colombia; Fundación Valle del Lili, Cali, Colombia

Pulmonary hypertension in patients with DTGA may suffer delayed definitive treatment and put them at risk of mortality; we present a case of high complexity in their management. The objective of this study was to analyze the case of a newborn with complex congenital heart disease, with severe pulmonary hypertension that develops complications with risk of death, and analyze the systematic management of their clinical condition and outcome. Presentation of clinical case with clinical history data, reports of echocardiography, cardiac catheterization, and surgical medical management are analyzed, with complications during their stay and the outcome at the time of discharge from the unit. Patient on admission, diagnosed with DTGA with intact ventricular septum, is evaluated by serial echocardiograms diagnosing systemic supra pulmonary hypertension, with a high risk of mortality, which does not make him a candidate for elective surgery. Different alternatives for medical management, such as use of nitric oxide, atrioseptostomy, PGE1, Sildenafil, and bosentan, are evaluated and after one month, Jatene surgery is carried out, with subsequent stabilization and improvement of signs of hypertension over time. The patient was suffering from complex congenital heart disease with a poor prognosis due to the presence of pulmonary hypertension which was very difficult to control, subject to several medical management strategies to compensate and subsequently be taken to surgery after a long wait, but in the end, stabilization was achieved and was able to be taken to definitive correction despite several secondary complications. In conclusion, patients with this type of congenital heart disease have in themselves a prognosis of considerable morbidity and mortality that, together with the development of pulmonary hypertension, may be exposed to a high probability of higher mortality, but they are the waiting and application of various medical management strategies those that ultimately allow us to finally deliver a definitive and effective surgical correction.

Launching the Colombian network of pulmonary arterial hypertension “HAPred.co”

Mauricio Orozco-Levi, Melissa Mogollón, Javier Fajardo, Alba Ramírez-Sarmiento, Rafael Conde, Héctor Ortega and Manuel Pacheco

Alejandro Londoño; the HAPred.co Network Investigators

Servicio de Neumología, Centro para el Cuidado de la Salud Respiratoria, Hospital Internacional de Colombia, Santander, Colombia; Universidad de Santander, Santander, Colombia; Universidad Industrial de Santander (UIS), Bucaramanga, Colombia; IPS Respiremos, Pereira, Colombia; Clínica CardioVid, Medellín, Colombia; Fundación Neumológica Colombiana, Bogotá, Colombia

There are no global epidemiological or clinical data in relation to incidence, prevalence, clinical-functional characteristics, access to treatments, or prognosis of pulmonary arterial hypertension (PAH) or Chronic Thromboembolic Pulmonary Hypertension (HPTEC) in Colombia. This implies a need for networking in order to define the clinical characteristics and needs that allow the design of country-specific strategies that allow the resolution of health problems related to PAH and HPTEC in Colombia. Since 2018, a multidisciplinary professional group conceived and made possible an initiative aimed at establishing a multidisciplinary, inclusive, cohesive, and non-profit National Network, which when sharing activities and competences of various degrees, defined activities of various types focused on patients of Pulmonary Hypertension of Colombia. This initiative received the name of the Colombian Network of Pulmonary Arterial Hypertension (HAPred.co). The main objectives of the Network are oriented to (1) characterize the problem of PAH in the country, (2) solve epidemiological, clinical, and economic questions, (3) develop research in own population, (4) normalize related knowledge with the diagnosis, treatment, and follow-up of PAH in the region, (5) promote fundraising for own scientific and academic activities, and (6) offer advice on particular problems of patients with PAH in Colombia. The consensus of the network HAPred.co essence was identified as an initiative framed within 10 particular characteristics that represent its own value in its vision: (1) it reflects a fundamentally academic intention aimed at practical translation; (2) it is of a purely voluntary participation, where conflicts of interest solely individual are ignored; (3) it is feasible, since its scope has the attitude and competencies of health personnel and associates who have agreed on the action; (4) it is of a relevant nature, since it promotes the application of the knowledge and standards of diagnosis and management of PAH in Colombia; (5) it is original, because there is no similar initiative in this area in Colombia; (6) it is pertinent, for trying to cover social, clinical, economic, and academic aspects of PAH in Colombia; (7) it is inclusive, since it fundamentally invites and promotes a transdisciplinary and multi-institutional nature; (8) it is interdependent, where all researchers and related centers share their experiences and promote common solutions; (9) it is directed by objectives, with the desire to be facilitator and operational with specific products that benefit the management of the disease in the country; (10) it is our group vision that the work in La Red will be representing an offer of particular and relevant value centered in Colombia, with expanded projection to Latin America in interaction with external referent groups. To date, a total of n = 503 registries of both incident and prevalent PAH cases have been identified as a result of 10 months of launching the initiative. Members of HAPRed.co and the “HEXAGON” model: the activities of HAPred.co focus on patients suffering from PAH. For this reason, we develop activities aimed at achieving a “hexagonal integration”. The centers of the hexagon are “the patients”, and each vertex represents the different actors in their care system: hospitals (IPS), payers (EPS), Logistics Operators, Patient Support Groups, Patient Associations, and Pharmaceutical Industries. For this reason, the Network is a cross-cutting initiative among medical specialties and invites the participation of representatives from each vertex so that the platform is viable and relevant in achieving patient benefits. Governance and management model of the HAPRed.co: since its inception, the Network has an Operational Core, which is responsible for centralizing and consolidating the contributions of all its members. There are three fundamental ways to participate in the actions of the Network: graduated (member of the common forum of meetings and actions), researcher (principal investigator or collaborator in research actions initiated by a third party), and member of the Operational Nucleus (collaborator in the same network management). During the elapsed time of management, public master documents have been generated in which collaboration strategies with potential financing entities (e.g. pharmaceutical industry, state entities) or agreements with international academic entities have been defined. The actions, governance model, and transdisciplinary inclusion policies of the HAPred.co were agreed upon. As such, it does NOT depend on a single institution, it is NOT a defined time strategy, it does NOT have a unique disciplinary orientation, and it does NOT depend on a single investigator or manager. For these reasons, we believe that La Red constitutes a platform for continuous action that allows the interests of multiple participants to be housed under the sole premise of offering useful elements and potential benefits to patients with PAH in Colombia.

Baseline risk evaluation predicts likelihood of clinical worsening: a sample size re-analysis from Freedom-EV

R.J. White, Youlan Rao, C.Q. Deng, Andrew Nelsen and Raymond L. Benza

University of Rochester Medical Center, Rochester NY, USA; United Therapeutics, Research Triangle Park, NC, USA; Allegheny Health Network, Pittsburgh, PA, USA

Multiparametric risk analyses like the Reveal 2 or French non-invasive (French) risk scores predict outcomes for patients with pulmonary arterial hypertension (PAH). Freedom-EV was a global, event-driven study of oral treprostinil in participants who had recently started monotherapy as treatment for PAH. In Freedom-EV, we prospectively planned to analyze the change in the French non-invasive risk score, and, post-hoc, we also analyzed the change in Reveal 2 score. For this post-hoc analysis, we hypothesized that participants who had clinical worsening (CW) events started with a higher baseline risk score than those who did not. We hypothesized that enriching a study selecting only those with higher risk scores would allow a smaller number of participants. We calculated the French and Reveal 2 risk scores for four groups: active treatment with CW events, active without events, placebo with events, and placebo without events. We used Fisher’s exact analysis to compare categorical French risk scores and a non-parametric ANCOVA to analyze continuous Reveal 2 scores. We re-analyzed sample size enrolling only participants with 0 or 1 French low-risk factors with an underlying hazard ratio of 0.625 (observed among higher risk participants in Freedom-EV) and a placebo median event time of 20 months. We assumed accrual during 36 months (total study duration 42 months) with a 19% active dropout rate and 10% placebo dropout. Results showed that both scores demonstrated statistically higher risk among those with CW events as compared to those without (Table 1). Our analyses suggest that we would have 90% power to demonstrate the same hazard ratio with 191 CW events and 430 higher risk participants (French score of 0 or 1 low-risk factors) as compared to the 205 CW events and 690 participants actually enrolled. In conclusion, for event-driven studies, enriching for those with higher baseline risk may be reasonable.

Table 1. ▪

	Active-CW event	Active-no event	Placebo-CW event	Placebo-no event
Baseline Reveal 2 Mediana (Min, Max)	7 (3, 13)	6 (2, 13)	7 (2, 12)	5 (1, 13)
N (for reveal)	85	252	120	213
Baseline No. of French low-risk factorsb
0 low risk	31/85 (36%)	54/252 (21%)	35/120 (29%)	24/214 (11%)
1 low risk	36/85 (42%)	76/252 (30%)	50/120 (42%)	60/214 (28%)
2 low risk	15/85 (18%)	87/252 (34%)	20/120 (17%)	74/214 (35%)
All three low risk	3/85 (4%)	35/252 (14%)	15/120 (12%)	56/214 (26%)

^aReveal two difference between groups, ANCOVA, p < 0.001.

^bFrench difference between groups, Fisher’s exact, p < 0.001.

Time course and prognostic significance of pulmonary artery pressure in highlanders. A prospective cohort study

T.M. Sooronbaev, M. Mademilov, B. Emilov, N. Marajapov, U. Sheraliev, M. Furian, S. Ulrich and K.E. Bloch

National Center for Cardiology and Internal Medicine, Bishkek, Kyrgyzstan; Pulmonary Division and Sleep Disorders Center, University Hospital of Zurich, Zurich, Switzerland

In a prospective cohort of Kyrgyz highlanders, we evaluated mean pulmonary artery pressure (mPAP) and clinical outcomes. Starting from the baseline measurements in 2012, annual follow-up examinations were carried out during summer time over the course of the study period (2012–2017). Life-long residents in the Aksay high-altitude plateau (Kyrgyzstan, altitude 3000–4000 msl) free of overt lung or cardiovascular disease and non-polycythemic were invited to undergo yearly echocardiography, clinical examinations, six-minute walking test, spirometry, and nocturnal cardiorespiratory monitoring. Deaths were recorded. Results showed that of the 165 participants, 71 (43%) were women. The mean age was 43 ± 12 (range: 17–75) years. In 2012, mPAP was 27.8 ± 3.8 mmHg. There was a significant increase of mPAP with age. The yearly increase in adjusted mPAP was 0.34 mmHg/year (95%CI: 0.29–0.40, P < 0.001), that exceeded the age related increase in lowlanders (0.1 mmHg/year increase in RV/RA, McQuillan Circulation, 2001). In univariable analysis, the current age, the baseline body mass index, the mean systemic arterial pressure, the mean nocturnal SpO₂, and the oxygen desaturation index (ODI) were significant predictors of the mPAP over the course of the study period, while the forced expiratory volume in 1 second (FEV1) was not a significant predictor. In multivariable analysis, only the current age and the BMI at baseline were significant predictors of the mPAP. During this five-year observation period, a total of six participants died. In Cox regression analysis, mPAP was a significant predictor of death in all participants. In conclusion, in the current cohort of highlanders, mean mPAP was higher than values reported for healthy lowlanders, and the yearly increase in adjusted mPAP was 0.34 mmHg/year. We found that the current age, the baseline BMI, the mean nocturnal SpO₂, and the ODI were significant predictors of the mPAP. Further studies are required to investigate the mechanisms linking higher values of mPAP with a greater risk of death. (This study was supported by OPO foundation, Zurich Lung League, Swiss Lung Foundation).

The anti-lung cancer drug (R)-Crizotinib predisposes and exacerbates pulmonary hypertension

Charifa Awada, Francois Potus, Eve Tremblay, Roxane Paulin, Steeve Provencher, Olivier Boucherat and Sebastien Bonnet

Pulmonary Hypertension and Vascular Biology Research Group of Quebec Heart and Lung Institute, Laval University, Quebec, Canada

Pulmonary arterial hypertension (PAH) is a vascular remodeling disease characterized by persistent elevation of pulmonary vascular resistance. The disease leads spontaneously to right heart failure and death. In 10% of cases, PAH is induced by drugs. Recently, PAH induced by chemotherapeutic agents such as RTK inhibitors (e.g. dasatinib) has been described. (R)-Crizotinib is an ALK/ROS1/MET inhibitor increasingly used for the treatment of some non-small cell lung cancer. Interestingly, (R)-Crizotinib has been shown to induce endothelial cells (ECs) dysfunction and is symptomatically associated with dyspnea and peripheral edema in some patients, which are central symptoms of PAH. We thus hypothesized that chronic administration of (R)-Crizotinib exacerbates and predisposes PAH. We found that daily oral administration of (R)-Crizotinib (100 mg/kg for two weeks) in Sugen/Hypoxia (Su/Hx)-challenged rats with established PAH significantly increased mortality rate. Compared to vehicle-treated (Su/Hx) rats, (R)-Crizotinib was associated with worsening of hemodynamic parameters (RVSP, mPAP, Sv, CO, and TPR measured by right heart catheterization; p < 0.05; n = 4–6). Histologically, (R)-Crizotinib enhanced pulmonary arteries (PA) wall thickness (EVG stain; p < 0.05), and increased accumulation of macrophage and fibrosis within the lungs and RV. In addition, we observed that pretreatment of rats with (R)-Crizotinib exaggerates the response to monocrotaline (MCT, 40 mg/kg) administration, as revealed by histological (increase in vascular remodeling) and hemodynamic measurements (mean PAP; TPR; CO) (p < 0.01; n = 7–10). In vitro, (R)-Crizotinib reduced MET and AKT activation (WB) in human PA ECs promoting endothelial dysfunction, (decreased proliferation (Ki67) and increased apoptosis (Annexin V). We documented for the first time that (R)-Crizotinib treatment predisposes and excacerbates PAH in animal models. This study could have major clinical relevance in the management of patients treated with (R)-Crizotinib or next-generation ALK/ROS1/MET inhibitors.

Assessment of right ventricular diastolic by tricuspid annular plane diastolic excursion and its prognostic value in patients with pulmonary arterial hypertension

Prashant Bobhate, Sylvia Colaco, Tanuja Karande and Snehal Kulkarni

Childrens’ Heart Centre, Kokilaben Dhirubai Ambani Hospital, Mumbai, India

Complete assessment of right heart function includes assessment of the right atrial and ventricular function. Right ventricular systolic function can be assessed by 2D echocardiogram using tricuspid annular plane systolic excursion (TAPSE) and right ventricular fractional area change (RVFAC). Right ventricular diastolic function is difficult to assess especially in pediatric patients. TAPSE has an upstropke and a downstroke. The displacement in the upstroke is used to measure the RV systolic function. The downstroke could be utilised for assessing the right ventricular diastolic function. Right ventricular diastolic function and its importance in the management of patients with pulmonary arterial hypertension is currently not established. In this study, we aimed to quantify the right ventricular diastolic function using TAPSE and establish its prognostic significance. We studied patients of PAH without shunt lesions referred to our centre. All patients underwent a detailed clinical examination, echocardiogram, cardiac catheterization, NT pro-BNP and six-minute walk test whenever feasible. All patients were regularly followed up. Clinical deterioration was defined as death, creation of Potts shunt or addition of prostacyclin analogues. Total right heart function was measured using TAPSE. Downstroke in TAPSE was broken down into two components, excursion occurring from the peak of the TAPSE to the beginning of p wave on ECG (TAPSE_Rv) and from the beginning of the p wave to the trough of TAPSE (TAPSE_RA). The percentage of TAPSE_RA (%TAPSE_RA) in relation to the entire TAPSE was calculated. We studied 48 children (17 F), median age 3 (range: 0.3–17) years, median BSA 0.56 (0.2–1.8) m². Echocardiogram was done on all patients at the time of initial presentation to the PAH clinic. Eight patients underwent Potts shunt, one was started on iloprost and there were six deaths. Median event-free survival was 2 (0.2–3.4) years. One- and 3-year event-free survival were 86 and 58% respectively. %TAPSE RA was significantly higher in patients with clinical deterioration (72 ± 6 vs. 34 ± 8), p ≤0.001 and correlated with higher NT-proBNP and lower six-minute walking distance. %TAPSE _RA of > 60% and RVFAC of < 25% were independently associated with event-free survival at three years (p = 0.02 and 0.03). In children with PAH, RV systolic and diastolic dysfunction are associated with poor outcomes. Diastolic dysfunction precedes systolic dysfunction in patients with PAH. %TAPSE_RA can be used as a marker of reliance on atrial contribution in maintaining cardiac output. Loss of this contribution can lead to clinical deterioration. Regular monitoring of %TAPSE_RA can identify the subset of patients who are prone to clinical deterioration and thus need closer follow-up and escalation of therapy.

Abernathy malformation: a potentially treatable cause of pulmonary hypertension

Prashant Bobhate, Sandeep Garg, Tanuja Karande, Pankaj K. Kasar and Snehal Kulkarni

Childrens’ Heart Centre, Kokilaben Dhirubai Ambani Hospital, Mumbai, India

Pulmonary arterial hypertension (PAH), is a progressive disease with fatal outcome. A detailed evaluation of patient with PAH is necessary before initiation of therapy. Congenital extrahepatic porto-systemic shunts (Abernathy malformation) are rare but often missed as a potential cause of PAH. If identified and closed, this can be an important cause of treatable PAH. We present a case series of patients with PAH and Abernathy malformation. This is a retrospective analysis of a single-centre experience of patients with PAH and Abernathy malformation. From June 2014 to August 2019, all patients referred at our centre for PAH undergo detailed evaluation including USG abdomen and portal venous Doppler. The children who were suspected to have portocaval abnormalities on USG underwent CT angiogram to confirm the diagnosis. Patients underwent closure of the shunt when feasible. Pre and post procedure functional class, echocardiogram and medication requirement was recorded for all patients. We encountered seven patients (median age = 13 years, range = 3.5–16 years) with PAH who on evaluation also had Abernathy malformation. Five out of seven patients presented with dyspnoea on exertion and two out of seven presented with syncope. All patients were diagnosed to have “idiopathic PAH” at outside centres and were referred to us for evaluation and management. All the children had moderate to severe PAH on initial echocardiography at our centre. Six out of seven patients were on dual pulmonary vasodilators (PDE5 inhibitors and ET receptor blockers) and one patient was on PDE5 inhibitor at presentation. On evaluation at our centre, six out of seven were diagnosed to have Abernathy malformation type 2 and one was diagnosed to have Abernathy malformation type 1 on USG abdomen and portal venous Doppler. Six out of seven patients underwent cardiac catheterization with balloon occlusion of the Abernathy malformation. At baseline, mean PA pressures were 56 ± 14 mmHg and pulmonary vascular resistance index (PVRi) of 6.7 ± 2 Woods units.m². At baseline, portal pressures were 8 ± 2 mmHg, there was an increase in portal pressures of 3 mmHg (range = 2–8 mmHg) after balloon occlusion of the lesion. Three out of six patients with type 2 malformation underwent device closure and one underwent surgical closure as the malformation was not suitable for device closure. Two patients were lost to follow-up. At follow-up, median duration of eight months (3–11 months), one patient had complete normalisation of PA pressure and is now off pulmonary vasodilators and the remaining two are on single drug (PDE5 inhibitor) with mild PAH on echocardiogram. There was a significant improvement in functional class IV vs. II (range I–II), p = 0.03) in all patients who underwent closure of the malformation. Abernathy malformation is a rare but potentially treatable cause of PAH in children. All patients with PAH should undergo USG abdomen and portal venous Doppler to identify and treat this disorder.