Pulmonary arterial hypertension – progress in understanding the disease and prioritising

strategies for drug development

Pavandeep Ghataorhe1, PhD; Christopher J. Rhodes1, PhD; Lars Harbaum1, MD; Mark Attard1, BM

BCh; John Wharton1, PhD; Martin R. Wilkins1, MD

Affiliations

1Department of Medicine, Imperial College London, Hammersmith Campus, Du Cane Road, London,

W12 0NN, UK

*corresponding author: m.wilkins@imperial.ac.uk, 020 3313 2049

Abstract

Pulmonary arterial hypertension (PAH), at one time a largely overlooked disease, is now the subject

of intense study in many academic and biotech groups. The availability of new treatments has

increased awareness of the condition. This in turn has driven a change in the demographics of PAH,

with an increase in the mean age of diagnosis. The diagnosis of PAH in more elderly patients has

highlighted the need for careful phenotyping of patients and for further studies to understand how

best to manage pulmonary hypertension associated with, for example, left heart disease.

The breadth and depth of expertise focused on unravelling the molecular pathology of PAH has

yielded novel insights, including the role of growth factors, inflammation and metabolic remodelling.

The description of the genetic architecture of PAH is accelerating in parallel, with novel variants,

such as those reported in potassium two pore domain channel subfamily K member 3 (KCNK3),

adding to the list of more established mutations in genes associated with bone morphogenetic

protein receptor type 2 (BMPR2) signalling. These insights have supported a paradigm shift in

treatment strategies away from simply addressing the imbalance of vasoactive mediators observed

in PAH towards tackling more directly the structural remodelling of the pulmonary vasculature.

Here we summarise the changing clinical and molecular landscape of PAH. We highlight novel drug

therapies that are in various stages of clinical development, targeting for example cell proliferation,

metabolic, inflammatory/immune and BMPR2 dysfunction and the challenges around developing

these treatments. We argue that advances in the treatment of PAH will come through deep

molecular phenotyping with the integration of clinical, genomic, transcriptomic, proteomic and

metabolomic information in large populations of patients through international collaboration. This

approach provides the best opportunity for identifying key signalling pathways, both as potential

drug targets and as biomarkers for patient selection. The expectation is that together these will

enable the prioritisation of potential therapies in development and the evolution of personalised

medicine for PAH.

Introduction

Pulmonary hypertension (PH) is a rare disorder with a global prevalence of 1% 1. It is defined by a

resting mean pulmonary artery pressure (mPAP) ≥25mmHg during right heart catheterisation 2. A

mPAP ≥25mmHg is an independent predictor of survival in a number of clinical presentations 3, 4, but

there is a continuum of risk with borderline PH, classified as mPAP between 19 and 24mmHg,

associated with increased hospitalisations and mortality 3, 5.

PH is classified into 5 main subgroups by international consensus, based on clinical features,

including the presence of a co-existing disease, haemodynamic measurements and, increasingly,

genetic information 6 (Table 1). This clinical classification has its limitations as it lacks the granularity

to tailor treatment according to individual patient phenotypes 7. Pulmonary arterial hypertension

(PAH) is the first main category and characterises a subgroup of patients with pre-capillary disease

(mPAP≥25mmHg and pulmonary arterial wedge pressure (PAWP) ≤ 15mmHg) and increased

pulmonary vascular resistance (PVR >3 Wood units). It is inclusive of congenital heart disease and

connective tissue disease but excludes chronic thromboembolic disease, hypoxic and interstitial lung

disease, and miscellaneous diseases which have their own categories 6. Patients with idiopathic

pulmonary arterial hypertension (IPAH) are the most common form of PAH and this is a diagnosis of

exclusion 6. PH presents most frequently in association with other diseases such as left heart disease

(PH-LHD) and chronic lung disease. PH-LHD is recognised as post-capillary pulmonary hypertension,

from backward transmission of high left-sided filling pressure, and is defined haemodynamically by a

mPAP ≥25 mmHg with PAWP >15 mmHg. A significant number of patients with PH-LHD have

increased PVR >3 Wood units and a diastolic pressure gradient ≥7 mmHg, suggesting a pre-capillary

component to their pulmonary hypertension

Clinical Classification

Haemodynamic Classification

1. Pulmonary arterial hypertension Pre-capillary1.1 Idiopathic1.2 Heritable

1.2.1 BMPR2 mutation1.2.2 ALK-1, ENG, SMAD9, CAV1, KCNK3 mutations1.2.3 Unknown

1.3 Drugs and toxins induced1.4 Associated with:

1.4.1 Connective tissue disease1.4.2 Human immunodeficiency virus (HIV) infection1.4.3 Portal hypertension1.4.4 Congenital heart disease1.4.5 Schistosomiasis

1’. Pulmonary veno-occlusive disease and/or pulmonary capillary haemangiomatosis1”. Persistent pulmonary hypertension of the newborn2. Pulmonary hypertension due to left heart disease Post-capillaryIncluding left ventricular systolic/diastolic dysfunction, valvular and congenital disease3. Pulmonary hypertension due to lung diseases and/or hypoxia Pre-capillaryIncluding obstructive, restrictive, interstitial and developmental lung disease4. Chronic thromboembolic pulmonary hypertension and other pulmonary artery obstructions Pre-capillary4.1 Chronic thromboembolic pulmonary hypertension4.2 Other pulmonary artery obstructions

5. Pulmonary hypertension with unclear and/or multifactorial mechanismsPre- & post-capillary

Table 1 – Updated clinical classification of pulmonary hypertension. Adapted from Simonneau et

al., 2013 and Galie et al., 2015 6, 8. Haemodynamic features of each subgroup are also shown. Pre-

capillary pulmonary hypertension is defined as mean pulmonary artery pressure (mPAP) ≥25mmHg

and pulmonary arterial wedge pressure (PAWP) ≤ 15mmHg, whilst post-capillary pulmonary

hypertension is mPAP ≥25mmHg and PAWP >15mmHg. BMPR2, bone morphogenetic protein

receptor, type 2; ALK-1, activin-like receptor kinase-1; ENG, endoglin; CAV1, caveolin-1.

A multi-centre US based registry for PAH (Registry to Evaluate Early and Long-Term Pulmonary

Arterial Hypertension Disease Management - REVEAL registry) indicates an incidence and prevalence

of 2.0 and 10.6 cases of PAH per million inhabitants respectively 9. Registries from the UK show

similar incidence and prevalence rates of 1.1 and 6.6 cases of PAH per million inhabitants

respectively 10.

The currently licensed therapies for PAH target the three main vasoactive pathways, namely -

prostacyclin-cAMP, nitric oxide-soluble guanylate cyclase-phosphodiesterase type 5 (cGMP-PDE5)

and endothelin 6. Despite improvement in patient symptoms and well-being with these agents,

mortality rates remain high (~65% survival at 5 years) 11 and there is a need for therapies targeting

alternative pathways that can reverse pulmonary vascular remodelling, inhibit disease progression

and improve survival.

The changing demographics and revisiting the haemodynamic classification

PAH is seen more commonly in women. The REVEAL registry records a 4:1 female: male ratio 12 and

72% of patients are female in a recent meta-analysis of 1550 patients with IPAH, hereditary PAH

(HPAH) or anorexigen-induced PAH 13. But the demographics of PAH are changing. Earlier registries

record the mean age at presentation as 36 years 14. Increasingly the diagnosis is made in the elderly

population, and current registries show a mean age between 50-65 years at diagnosis 6, 10, 12, 15.

Elderly patients (over 75 years) diagnosed with IPAH have less gender bias than younger patients

and a higher mortality, following adjustment for age 15. Older patients have more co-morbidities,

with an increased number of risk factors for LHD. This has led to the use of terms such as ‘typical

IPAH’, which refers to patients who have fewer than 3 risk factors for LHD, and ‘atypical IPAH’, who

have 3 or more risk factors for LHD 16.

A comparison of patients with ‘typical IPAH’, ‘atypical IPAH’ and pulmonary hypertension associated

with heart failure and preserved ejection fraction (HFpEF) has shown similar mPAP, cardiac output

and long-term survival 16. One view expressed is that there is a spectrum of disease ranging from

PAH to HF-pEF with combined pre-capillary and post-capillary (CpC-PH) bridging the two 6, 17. The

lungs of CpC-PH patients exhibit histological evidence of pulmonary vascular remodelling similar to

PAH 18, but, in general, patients with PH-LHD are not responsive to therapies developed for PAH, and

show a higher incidence of side effects. Current guidelines discourage the use of PAH therapies in

patients with HF-pEF and a greater understanding of the molecular drivers of PH in these patients

will help develop better treatment strategies 16.

Pathophysiology – recognising proliferation, inflammation and metabolic remodelling

PAH is characterised by the narrowing of small pulmonary arteries through vasoconstriction,

thrombosis, and proliferation and remodelling of the vessel wall 19, 20. This includes characteristic

neointimal and plexiform lesions, and medial hypertrophy, propagated by the proliferative and anti-

apoptotic phenotypes of pulmonary endothelial cells, smooth muscle cells, fibroblasts and

myofibroblasts 21. There is also increasing evidence of a transition of pulmonary artery endothelial

cells to smooth muscle like mesenchymal cells (endothelial-to-mesenchymal transitioning) which can

be driven by dysfunctional bone morphogenic protein receptor type II (BMPR2) signalling 22-24, the

predominant cause of hereditary PAH 25, 26. Occlusion of the lumen in small distal pulmonary arteries

leads to increased PVR, and subsequently increased right ventricular (RV) afterload and RV failure 6,

27.

Given the pronounced structural remodelling of diseased vessels, it is surprising that little attention

has been paid to the potential growth factors involved until relatively recently. The idea that the

vascular lesions of PAH exhibit many of the traits of cancer (abnormal and uncontrolled cell growth,

angioneogenesis, evasion of apoptosis, self-sufficiency in growth signals and insensitivity to anti-

growth signals) and may be a neoplastic angioproliferative disorder took a while to challenge the

prevailing opinion that PAH was the consequence of chronic and sustained vasoconstriction 28, 29. But

the ‘cancer paradigm’ concept is now firmly established and frames the investigation of many new

drug targets and the possibility of re-purposing anti-cancer drugs 30.

While there is no evidence of constitutively active growth factors in PAH, patient-derived pulmonary

vascular cells show an increased proliferative response to mitogenic stimuli, including fetal calf

serum, fibroblast growth factor-2 (FGF-2), epidermal growth factor (EGF), vascular endothelial

growth factor and platelet-derived growth factor (PDGF), and are less sensitive to apoptosis

induction by serum deprivation 30. Such hyper-proliferative potential could be explained by

overexpression and/or activation of receptor tyrosine kinases, including EGF, FGF and PDGF

receptors. A clinical trial to investigate the efficacy of the PDGFR inhibitor, imatinib, in PAH suggests

that some patients may respond and benefit from this approach but the development programme

has been halted because of side effects and concerns about safety 31-33. There remains considerable

interest in further targeting growth factors, including PDGF, in PAH.

A role for inflammation in PAH was recognised nearly 25 years ago 34. Histology of diseased vessels

show a significant influx of inflammatory cells, including macrophages and lymphocytes. Beyond

increased perivascular immune cell accumulation and intravascular infiltration, circulating levels of

certain cytokines and chemokines are abnormally elevated 35. These include interleukin (IL)-1β, IL-6,

IL-8, monocyte chemoattractant protein -1, fractalkine, chemokine ligand 5/regulated on activation

normal T cell expressed and secreted (CCL5/RANTES) and tumor necrosis factor -α. More recently

lymphoid neogenesis has been implicated in the vascular remodelling process in the lungs of PAH

patients 36. Whether inflammation is a key driver of PAH or part of the healing process merits pause

for thought, but the evidence is in favour of the former; some of these cytokines and chemokines

correlate with a worse clinical outcome in PAH patients and may serve as biomarkers of disease

progression. A definitive answer awaits an anti-inflammatory intervention that changes the course of

the disease for the better.

Metabolic remodelling is also a feature shared by vascular cells in PAH and cancerous cells 37-41.

Central to this is energy metabolism in mitochondria 38. Proliferating cells exhibit a shift to aerobic

glycolysis, mediated predominantly by pyruvate dehydrogenase kinase (PDK), which inhibits

pyruvate dehydrogenase 41. This has been reported in both the pulmonary vasculature and the right

ventricle in PAH 42. Evidence of increased metabolites from the tricarboxylic acid (TCA) cycle and

fatty acid oxidation, and altered arginine and sphingosine pathways have been found from mass

spectrometry analysis of lung tissue from PAH patients 43, 44. Restoration of glucose oxidation, either

directly or by exploiting the Randle cycle to decrease fatty acid oxidation and glutaminolysis,

provides a potential therapeutic target for patients with PAH (Figure 1). As an exemplar of this

approach, administration of dichloroacetate (DCA), to directly inhibit PDK, improves both pulmonary

vascular disease and right ventricular function in animal models of PAH 45-48, and is currently under

investigation in patients with PAH (http://www.clinicaltrials.gov; NCT01083524).

Figure 1 – Mitochondrial energy metabolism. The three main energy pathways in the mitochondria

are shown including fatty acid/beta oxidation (green), glucose oxidation/aerobic glycolysis (blue) and

glutaminolysis (purple) which feed into the tricarboxylic acid (TCA) cycle (pink). Acetyl-coA produces

*citrate which is transported from the mitochondria to the cytosol where it inhibits

phosphofructokinase (PFK) and glucose oxidation (Randle effect). Increased pyruvate dehydrogenase

kinase (PDK) inhibits pyruvate dehydrogenase (PDH) and glucose oxidation, leading to aerobic

glycolysis and the generation of lactate (Warburg effect). Dichloroacetate (DCA) directly inhibits PDK

to restore glucose oxidation. Drugs such as ranolazine (RAN) and trimetazidine (TMZ) inhibit fatty

acid oxidation. *citrate is transported from the mitochondria to the cytosol where it inhibits

phosphofructokinase (PFK). GLUT1, glucose transporter 1; glucose-6P, glucose 6 phosphate; HK1/2,

hexokinase 1/2; CoA, co-enzyme A; SOD2, superoxide dismutase-2 ; HIF-1 α, hypoxia inducible

factor-1α; Ca2+, calcium; CPT1/2, carnitine palmitoyltransferase 1/2; FATP1, fatty acid transport

protein 1.

Several other metabolic pathways have been implicated in the pathobiology of pulmonary arterial

hypertension, including steroid 49, arginine 44, 50, 51, polyamine 44, 52, 53, tryptophan 54-56 and sphingolipid

metabolism 57. Metabolic profiling of patients is an active area of research with increased circulating

acylcarnitines 58, 59 and modified nucleosides originating from transfer RNAs seen in patients with

PAH and linked to survival 58.

Genetic susceptibility to pulmonary arterial hypertension

The observation that mutations in BMPR2 increased susceptibility to PAH came from family studies

reported in 2000 60, 61. A recent meta-analysis of 1550 patients with idiopathic, heritable or

anorexigen forms of PAH found BMPR2 mutations in 29% of participants 13. Mutations were seen in

82% of patients with a family history and in 17% of cases with no known family history of the

disease. BMPR2 mutation carriers had an earlier onset of disease and higher mPAP and higher PVR.

They also had poorer outcomes related to survival and transplantation 13. Pathogenic mutations have

been found throughout the BMPR2 gene, including key regions such as the kinase domain 62. Despite

the importance of BMPR2 in the pathobiology of PAH, disease penetrance in carriers is only ~20%,

indicating that other genetic and/or environmental factors are required for development of the

phenotype.

BMPR2 is a receptor for multiple bone morphogenic proteins and acts to suppress vascular smooth

muscle cell growth through the intracellular Smad and LIM (Lin-11, Islet-1, Mec-3) kinase pathways 63. Highlighting the significance of this pathway in PAH, mutations have been reported (although less

commonly) in other genes in the BMP/TGF (transforming growth factor) beta signalling, including

genes encoding activin A receptor type II-like kinase-1 (ALK1/ACVRL1), endoglin (ENG), mothers

against decapentaplegic (SMAD) -4 and SMAD8 25, 26. Exome sequencing has also led to the

identification of other susceptibility genes, including caveolin-1 (CAV1) and potassium two pore

domain channel subfamily K member 3 (KCNK3) 64, but mutations in non-BMPR2 genes account for <

5% of PAH cases 65.

There is interest in identifying common polymorphisms that could represent a ‘second hit’,

influencing the presentation and severity of the PAH. For example, variants in the non-coding

promoter region of the prostacyclin synthase (PGIS) gene may be protective in the development of

PAH in unaffected BMPR2 carriers 66. Mutations in genes such as potassium voltage-gated channel

sub-family A Member 5 (KCNA5) or in the non-coding regions of the BMPR2 gene also have the

potential to affect the onset and severity of the disease process 67-70. In addition, variants in

endostatin (Col18a1) 71 and the endothelin-1 pathway gene GNG2 72 are associated with clinical

outcomes in PAH and may influence the response to treatment. Studies in large populations of

IPAH/HPAH patients are underway, such as the UK National Institute of Health Research Biomedical

Research Centres Inherited Diseases Genetic Evaluation (BRIDGE) consortium and US National

Biological Sample and Data Repository for PAH, to assess the prevalence of both common and rare

variants in PAH 73.

The right ventricular (RV) – pulmonary circulation as a cardiovascular unit

RV function determines survival in PAH 6, 27, 74. Initially, RV hypertrophy compensates for the

increased pressure afterload from raised pulmonary vascular resistance but the chronic elevation of

pressure-load leads to RV ischaemia, impaired RV ejection fraction and RV failure 75. In addition,

measures of right ventricular-arterial coupling, taking into account afterload, predict survival in

patients with PAH 76. The development of RV failure can vary between patients, despite having

similar degrees of RV hypertrophy and pulmonary pressures, with the RV progressively dilating and

decompensating more rapidly in some patients than others 77-79. Understanding the molecular basis

for adaptation of the RV, predicting RV dysfunction clinically and targeting it therapeutically form a

key aspect of the management and treatment of PAH.

Circulating biomarkers – providing insight into the disease and tools for diagnosis and monitoring

There remains considerable interest in identifying accessible non-invasive biomarkers that will assist

the clinical management of PAH. Regular clinical assessment is required to evaluate disease

progression, response to therapy and prognosis. This includes using multiple clinical parameters and

the application of risk equations, although these were designed to gauge prognosis at diagnosis and

cannot predict outcomes for individual patients 80. Haemodynamic measurements, both at baseline

(diagnosis) and during follow-up, are established predictors of disease severity and survival in PAH

patients, with right atrial pressure, cardiac index and mixed venous oxygen saturation being the

most robust haemodynamic indices of right ventricular function and prognosis 81-84. However,

patients with PAH do not often receive regular follow-up cardiac catheterisations due to the invasive

nature of the procedure, and so non-invasive markers that predict disease progression and survival

are of key importance.

World Health Organisation functional class (WHO-FC) is a predictor of survival in PAH patients 85, 86

and worsening WHO-FC is a marker of disease progression 83. Six minute walk distance (6MWD) is

readily measurable at a clinic visit and is a prognostic indicator in PAH 85, but it’s limited by the effect

of multiple confounding factors such as co-morbidities and age. It remains the most commonly used

clinical trial endpoint in PAH although a meta-analysis of trial data showed a change in 6MWD does

not predict disease progression and survival 87. Both measures lack complete objectivity and hence

may differ between centres and for reasons independent of PH severity.

Measurements of RV function derived from imaging modalities such as cardiac MRI (CMR) or

echocardiography predict survival in PAH patients 79, 88-91. Although many patients undergo CMR at

diagnosis, it is often not repeated due to the expense of the procedure. There remains scope for a

robust and readily measurable circulating marker to predict survival in PAH.

The most widely used and accepted circulating biomarker is N-terminal pro-brain natriuretic peptide

(NT-proBNP) 6. It is of limited use as a diagnostic marker as it lacks disease specificity, but has value

as a prognostic marker as circulating levels, particularly changes in circulating levels within an

individual, reflect disease progression and functional and haemodynamic impairment in PAH

patients 83, 92-95.

A wide range of other circulating factors have been investigated for risk stratification in PAH. Studies

have reported on markers of inflammation (e.g. C-reactive protein, interleukin-6), renal impairment

(e.g. creatinine, cystatin 2), liver dysfunction (e.g. bilirubin), vascular remodelling (e.g. angiopoetin-

2), myocardiocyte strain (e.g. soluble suppression of tumorigenicity - ST2), iron deficiency (e.g. red

cell distribution width - RDW) and microRNAs (e.g. microRNA-150) 94, 96-102 6, 103-106. The possibility that

factors that report on different components of the pathology of PAH might be combined to better

effect merits careful evaluation.

The availability of high-throughput platform technologies has allowed broad unbiased screens of

biological fluids for molecules related to the disease. One such approach has been the use of

metabolomics. Nuclear magnetic resonance spectroscopy and mass spectrometry allows the

simultaneous assessment of thousands of low molecular weight metabolites from lung tissue,

plasma and urine samples. A small study of 20 PAH patients and controls used an untargeted

metabolomics platform and showed changes in circulating metabolites such as increased lactate,

free fatty acids and glutamine in PAH patients, supporting a shift to aerobic glycolysis 107. A targeted

analysis of 105 circulating plasma metabolites in PAH, primarily amino acids, nucleosides and their

derivatives, showed abnormal levels of tryptophan, purine and TCA cycle metabolites correlated to

haemodynamic measures 56. A recent study of 1416 circulating metabolites in PAH showed evidence

of increased circulating modified nucleosides (N2,N2-dimethylguanosine, N1-methylinosine), TCA

cycle intermediates (malate, fumarate), glutamate, fatty acid acylcarnitines and polyamine

metabolites and decreased levels of steroids, sphingomyelins and phosphatidylcholines

characteristic of patients with PAH 58. Changes in an individual patient’s metabolite levels over time

were also associated with improved survival and patients defined as vasoresponders (who have

excellent outcomes on calcium channel blocker therapies) demonstrated metabolic profiles more

similar to healthy controls than other patients. Further work is needed to better understand how

these measurements may have clinical utility but in addition to monitoring the disease, this

approach can provide insight into novel therapeutic pathways. For example, targeting alterations in

energy metabolism in PAH or correcting translational regulation in PAH may provide future

therapeutic options in PAH 58.

The treatment of PAH – implementing a paradigm shift in therapy

Patients who retain vasoreactivity (>90%) to an acute vasodilator challenge show marked

improvements in quality of life and survival on long term oral calcium channel blocker therapy 6, 108.

The majority of patients are not ‘vasoresponders’ and commence targeted treatments for PAH,

alongside supportive treatments such as diuretics, focused on pharmacologically regulating the

activity of 3 vasoactive signalling pathways – the prostacyclin-cAMP, nitric oxide- cGMP-PDE5 and

endothelin pathways 6, 109, 110.

Guidelines for the use of these expensive therapies are revised at regular intervals to take account of

new treatments (e.g. soluble guanylate cyclase stimulators) 111 and new evidence. The latest

guidelines are available online 6 and the next likely revision will follow a meeting of experts in the

field in February 2018 (http://wsph2018.com). The current debate is whether to start with dual

therapy (a PDE5 inhibitor and endothelin receptor antagonist) or whether to proceed step wise,

starting with one and adding or replacing with the other. The nuance on this is whether the choice of

agents in the combination matter – given that the trial evidence of benefit comes from the

combination of tadalafil and ambrisentan 112.

The first generation endothelin receptor antagonists and PDE5 inhibitors were approved on the basis

of an improvement in 6MWD. More recent pivotal clinical trials have used a composite endpoint

that includes time to clinical worsening and death. There is some evidence from meta-analyses and

these later studies that targeted treatments improve survival. But the need to arrest and preferably

reverse the course of the disease remains a therapeutic challenge. One approach has been to initiate

treatment with three agents at once – prostacyclin, endothelin receptor antagonist and PDE5

inhibitor – to achieve a rapid reduction in pulmonary artery pressure and relieve the RV workload in

patients with severe disease. Early data look very encouraging 113. But although the licensed

therapies have some effect on vascular cell proliferation in vitro, there remains a keen interest in

identifying drugs which target the structural remodelling with greater potency. The results to date

with tyrosine kinase inhibitors, statins and serotonin antagonists have disappointed 114.

Although the translation of preclinical studies to patients is a significant challenge 114, 115, there is a

long list of potential new therapies. While many are still undergoing preclinical investigation, several

are currently in phase II clinical trials. These include compounds that target mitochondrial and

metabolic dysfunction (dichloroacetate, bardoxolone methyl), inflammation and immunity

(rituximab, tocilizumab, and ubenimex), BMPR2 signalling (tacrolimus), and iron deficiency (ferinject)

(Figure 2) 30, 116-118. Pulmonary artery denervation is also being investigated as a potential

interventional therapy 117 and recent advances in epigenetics, stem cells and gene editing offer

future translational advances in the treatment of PAH 73, 119.

Tryptophan hydrolase 1 inhibitor

Modulator of Nrf2 and NFkBPyruvate dehydrogenase inhibitor

FAC1 inhibitor

Phase I

Phase II

Phase III

Marketed

BosentanAmbrisentan

Macitentan

Epoprostenol

TreprostinilSelexipag

SildenafilTadalafil

Riociguat

MAP3 kinase inhibitor

Leukotriene A4 hydrolase inhibitorInterleukin-6 receptor inhibitor

mTOR inhibitor

Calcineurin inhibitor

Anti-CD20

3-Ketoacyl-CoA-thiolase inhibitor

Sodium current blocker

Oestrogen modulatorsUrosuric acid agent

Metabolic syndrome agentsSSRI

Aldosterone antagonist

NO supplementationIron supplementation

Cell therapy

Figure 2 – Licensed drugd and pathways under clinical investigation for the treatment of

pulmonary arterial hypertension. Nrf2, nuclear factor (erythroid-derived 2)-like-2 factor; NF-κB,

nuclear factor kappa-light-chain-enhancer of activated B cells; mTOR, mechanistic target of

rapamycin; NO, nitric oxide; SSRI, selective serotonin reuptake inhibitor.

The wide range of potential new treatments coupled to the shortage of patients for clinical trials has

created a problem and the need to prioritise treatments for investigation. To address this, we must

turn to ‘omic’ technologies and the concept of personalised medicine.

The application of ‘Omic’ technologies to pulmonary hypertension

It is widely appreciated that PAH, even IPAH, is a heterogeneous condition. Patients with similar

clinical manifestations vary markedly in their outcomes and responses to therapy. The clinical

classification is a blunt tool for selecting appropriate treatment and we need to do better. A systems

approach is required to understand the complex genetic, epigenetic and environmental influences

and the interactions of molecular pathways that underlie patient differences. The integration of data

obtained from genomics, transcriptomics, proteomics and metabolomics combined with clinical

phenotype information offers the potential of a comprehensive overview of the molecular basis of

the disease process. This ‘deep molecular phenotype’ provides the granularity to identify key

signalling pathways that are altered in the disease and assess potentially ‘druggable’ targets. It also

provides biomarkers that inform treatment options by predicting an individual’s response to therapy

(see Figure 3) and/or a means of monitoring response 73, 120, 121.

Patient 1 Patient 2 Patient 3 Patient 4

Drugs indicated: H J K L

PositiveNegative

Personalised medicine: prioritising drug selection based on -omics markers

Gene AGene family B

Gene cluster C

Protein DProtein cluster EProtein family F

Metabolite XMetabolite cluster Y

Metabolite class Z

Figure 3 – Personalised medicine: prioritising drug selection based on ‘omics’ markers.

Combinations of integrated biomarkers from ‘omics’ platforms which predict an individual’s

response to therapy could be used to guide the selection of drugs in a personalised medicine

approach. In the example above, 4 patients with similar clinical profiles could be prescribed different

clinically approved drugs (H, J, K, and L) based on the measurements of genes, proteins and

metabolites previously established to predict the response to each therapy.

Another area in which ‘omics’ analyses have great potential utility is in the identification of drugs,

even repurposing of drugs approved for other conditions 122, 123. A disease signature, for example

changes in gene and/or protein expression levels in patient tissue samples, can be compared with

the effects of libraries of drugs on gene and protein expression in relevant model systems (animal

and cell models). Drugs that restore or rebalance the pattern of gene and protein expression in these

model systems would then be candidates for PAH therapies (Figure 4).

Patient 1 Patient 2 Patient 3 Patient 4

Drugs indicated: H,J,K H,P,K J,P,K H,K,Q

PositiveNegative

Gene AGene family B

Gene cluster C

Protein DProtein cluster EProtein family F

Metabolite XMetabolite cluster Y

Metabolite class Z

Disease signature

Drugs’ effects in models1 2 3 4

Drug 2 selected

A

B

Re-purposing drugs by matching effects to disease signatures

Personalised medicine: prioritising drug selection based on -omics markers

Gene AGene family B

Gene cluster C

Protein DProtein cluster EProtein family F

Metabolite XMetabolite cluster Y

Metabolite class Z

Figure 4 – Re-purposing drugs by matching effects of disease signatures. Matching disease

signature profiles to the effects of drugs on those profiles in model systems could facilitate

identification and re-purposing of drugs approved for other conditions. In the example above, a

disease signature based on gene, protein and metabolite expression was established in samples

from a diseased population e.g. pulmonary arterial hypertension. This was compared to the effects

of 4 drugs on the same genes, proteins and metabolites in model systems. The profile of drug 2

influences the ‘disease signature’, restoring levels towards that seen in health, and could be selected

for further study as a treatment for the PAH.

Key to the success of this approach is access to clinically characterised patient cohorts linked to well-

curated sample biobanks. This has driven expert centres to collaborate at both the national and

international level to share protocols and harmonise data collection to power meaningful analyses. It

has also stimulated calls for large clinical trials to collect samples at baseline and study end, as these

sizeable cohorts of well-phenotyped patients defined by clear study entry criteria provide a precious

resource for ‘omics’ analyses to characterise the molecular makeup of responders and non-

responders to drug treatment.

Even so, the integration of multiple ‘omics’ datasets remains a challenge 124, 125. While several

software tools are available with large reference databases of molecular interactions, they have

limitations; in particular, incomplete biological pathway annotation 125. Exome data are better

annotated than whole genome sequence data and the regulation of protein levels is better

understood than protein-protein interactions, while epigenetic pathways, post-translation protein

modifications and metabolome data represent another level of complexity 124. The computational

analysis of multiple datasets and modelling of the dynamic interactions of genes, proteins and

metabolites through networks such as the Human Interactome Project

(http://interactome.dfci.harvard.edu/H_sapiens/) allows for prioritisation and identification of key

molecules 126-129. For example, a network built around pathogenic pathways in PAH has been used to

prioritise microRNAs, highlighting microRNA-21 which regulates Rho/Rho-kinase and BMP signalling 130. This approach can be applied to other ‘omics’ datasets to prioritise pathogenic targets in PAH.

Conclusions

The management of patients with PAH has advanced considerably over the past 25 years, with

improvements in functional status, quality of life and survival. In addition to their therapeutic

effects, drugs are powerful tools for dissecting disease mechanisms; responders and non-responders

to a given treatment have a story to tell. Coupled with a deeper understanding of the molecular

pathology of the disease process, the clinical view of PAH will continue to evolve. The idea that

molecular profiling of large cohorts of PAH patients will provide deeper phenotypic characterisation

of patients and a richer base from which to develop and target new signalling pathways tailored to

each patient’s needs has momentum. Delivery on this strategy will depend on international

collaboration to give sufficient power for network analysis to give confidence in the results. Success

will inform and likely shorten drug development by enabling patient selection for clinical trials and

monitoring response to treatment. It would be a ‘win-win’ scenario.

Acknowledgements

CJR is supported by a British Heart Foundation Intermediate Basic Science Research Fellowship

(FS/15/59/31839). PG and MA are supported by a grant from the Wellcome Trust – GSK Clinical

Fellowship Training Programme. MRW is supported by a British Heart Foundation programme grant

(RG/10/16/28575).

References

1. Hoeper MM, Humbert M, Souza R, Idrees M, Kawut SM, Sliwa-Hahnle K, Jing ZC and Gibbs JS. A global view of pulmonary hypertension. The lancet Respiratory medicine. 2016;4:306-22.

2. Hoeper MM, Bogaard HJ, Condliffe R, Frantz R, Khanna D, Kurzyna M, Langleben D, Manes A, Satoh T, Torres F, Wilkins MR and Badesch DB. Definitions and diagnosis of pulmonary hypertension. J Am Coll Cardiol. 2013;62:D42-50.

3. Maron BA, Hess E, Maddox TM, Opotowsky AR, Tedford RJ, Lahm T, Joynt KE, Kass DJ, Stephens T, Stanislawski MA, Swenson ER, Goldstein RH, Leopold JA, Zamanian RT, Elwing JM, Plomondon ME, Grunwald GK, Baron AE, Rumsfeld JS and Choudhary G. Association of Borderline Pulmonary Hypertension With Mortality and Hospitalization in a Large Patient Cohort: Insights From the Veterans Affairs Clinical Assessment, Reporting, and Tracking Program. Circulation. 2016;133:1240-8.

4. Aronson D, Eitan A, Dragu R and Burger AJ. Relationship between reactive pulmonary hypertension and mortality in patients with acute decompensated heart failure. Circ Heart Fail. 2011;4:644-50.

5. Maron BA and Abman SH. Focusing on Developmental Origins and Disease Inception for the Prevention of Pulmonary Hypertension. Am J Respir Crit Care Med. 2016;195:292-301.

6. Galie N, Humbert M, Vachiery JL, Gibbs S, Lang I, Torbicki A, Simonneau G, Peacock A, Vonk Noordegraaf A, Beghetti M, Ghofrani A, Gomez Sanchez MA, Hansmann G, Klepetko W, Lancellotti P, Matucci M, McDonagh T, Pierard LA, Trindade PT, Zompatori M and Hoeper M. 2015 ESC/ERS Guidelines for the diagnosis and treatment of pulmonary hypertension: The Joint Task Force for the Diagnosis and Treatment of Pulmonary Hypertension of the European Society of Cardiology (ESC) and the European Respiratory Society (ERS): Endorsed by: Association for European Paediatric and Congenital Cardiology (AEPC), International Society for Heart and Lung Transplantation (ISHLT). European Respiratory Journal. 2015;46:903-75.

7. Dweik RA, Rounds S, Erzurum SC, Archer S, Fagan K, Hassoun PM, Hill NS, Humbert M, Kawut SM, Krowka M, Michelakis E, Morrell NW, Stenmark K, Tuder RM, Newman J and Phenotypes ATSCoPH. An official American Thoracic Society Statement: pulmonary hypertension phenotypes. Am J Respir Crit Care Med. 2014;189:345-55.

8. Simonneau G, Gatzoulis MA, Adatia I, Celermajer D, Denton C, Ghofrani A, Gomez Sanchez MA, Krishna Kumar R, Landzberg M, Machado RF, Olschewski H, Robbins IM and Souza R. Updated clinical classification of pulmonary hypertension. J Am Coll Cardiol. 2013;62:D34-41.

9. McGoon MD, Benza RL, Escribano-Subias P, Jiang X, Miller DP, Peacock AJ, Pepke-Zaba J, Pulido T, Rich S, Rosenkranz S, Suissa S and Humbert M. Pulmonary arterial hypertension: epidemiology and registries. J Am Coll Cardiol. 2013;62:D51-9.

10. Ling Y, Johnson MK, Kiely DG, Condliffe R, Elliot CA, Gibbs JS, Howard LS, Pepke-Zaba J, Sheares KK, Corris PA, Fisher AJ, Lordan JL, Gaine S, Coghlan JG, Wort SJ, Gatzoulis MA and Peacock AJ. Changing demographics, epidemiology, and survival of incident pulmonary arterial hypertension: results from the pulmonary hypertension registry of the United Kingdom and Ireland. Am J Respir Crit Care Med. 2012;186:790-6.

11. Farber HW, Miller DP, Poms AD, Badesch DB, Frost AE, Muros-Le Rouzic E, Romero AJ, Benton WW, Elliott CG, McGoon MD and Benza RL. Five-Year outcomes of patients enrolled in the REVEAL Registry. Chest. 2015;148:1043-54.

12. Badesch DB, Raskob GE, Elliott CG, Krichman AM, Farber HW, Frost AE, Barst RJ, Benza RL, Liou TG, Turner M, Giles S, Feldkircher K, Miller DP and McGoon MD. Pulmonary arterial hypertension: baseline characteristics from the REVEAL Registry. Chest. 2010;137:376-87.

13. Evans JD, Girerd B, Montani D, Wang XJ, Galie N, Austin ED, Elliott G, Asano K, Grunig E, Yan Y, Jing ZC, Manes A, Palazzini M, Wheeler LA, Nakayama I, Satoh T, Eichstaedt C, Hinderhofer K, Wolf M, Rosenzweig EB, Chung WK, Soubrier F, Simonneau G, Sitbon O, Graf S, Kaptoge S, Di Angelantonio E, Humbert M and Morrell NW. BMPR2 mutations and survival in pulmonary arterial hypertension: an individual participant data meta-analysis. The lancet Respiratory medicine. 2016;4:129-37.

14. Rich S, Dantzker DR, Ayres SM, Bergofsky EH, Brundage BH, Detre KM, Fishman AP, Goldring RM, Groves BM, Koerner SK and et al. Primary pulmonary hypertension. A national prospective study. Annals of internal medicine. 1987;107:216-23.

15. Hoeper MM, Huscher D, Ghofrani HA, Delcroix M, Distler O, Schweiger C, Grunig E, Staehler G, Rosenkranz S, Halank M, Held M, Grohe C, Lange TJ, Behr J, Klose H, Wilkens H, Filusch A, Germann M, Ewert R, Seyfarth HJ, Olsson KM, Opitz CF, Gaine SP, Vizza CD, Vonk-Noordegraaf A, Kaemmerer H, Gibbs JS and Pittrow D. Elderly patients diagnosed with idiopathic pulmonary arterial hypertension: results from the COMPERA registry. International journal of cardiology. 2013;168:871-80.

16. Opitz CF, Hoeper MM, Gibbs JS, Kaemmerer H, Pepke-Zaba J, Coghlan JG, Scelsi L, D'Alto M, Olsson KM, Ulrich S, Scholtz W, Schulz U, Grunig E, Vizza CD, Staehler G, Bruch L, Huscher D, Pittrow D and Rosenkranz S. Pre-Capillary, Combined, and Post-Capillary Pulmonary Hypertension: A Pathophysiological Continuum. J Am Coll Cardiol. 2016;68:368-78.

17. Leopold JA. Biological Phenotyping of Combined Post-Capillary and Pre-Capillary Pulmonary Hypertension: Focus on Pulmonary Vascular Remodeling. J Am Coll Cardiol. 2016;68:2537-2539.

18. Chen Y, Guo H, Xu D, Xu X, Wang H, Hu X, Lu Z, Kwak D, Xu Y, Gunther R, Huo Y and Weir EK. Left ventricular failure produces profound lung remodeling and pulmonary hypertension in mice: heart failure causes severe lung disease. Hypertension. 2012;59:1170-8.

19. Tuder RM. Pulmonary vascular remodeling in pulmonary hypertension. Cell Tissue Res. 2016.

20. Tuder RM, Stacher E, Robinson J, Kumar R and Graham BB. Pathology of pulmonary hypertension. Clinics in chest medicine. 2013;34:639-50.

21. Stacher E, Graham BB, Hunt JM, Gandjeva A, Groshong SD, McLaughlin VV, Jessup M, Grizzle WE, Aldred MA, Cool CD and Tuder RM. Modern age pathology of pulmonary arterial hypertension. Am J Respir Crit Care Med. 2012;186:261-72.

22. Ranchoux B, Antigny F, Rucker-Martin C, Hautefort A, Pechoux C, Bogaard HJ, Dorfmuller P, Remy S, Lecerf F, Plante S, Chat S, Fadel E, Houssaini A, Anegon I, Adnot S, Simonneau G, Humbert M, Cohen-Kaminsky S and Perros F. Endothelial-to-mesenchymal transition in pulmonary hypertension. Circulation. 2015;131:1006-18.

23. Hopper RK, Moonen JR, Diebold I, Cao A, Rhodes CJ, Tojais NF, Hennigs JK, Gu M, Wang L and Rabinovitch M. In Pulmonary Arterial Hypertension, Reduced BMPR2 Promotes Endothelial-to-Mesenchymal Transition via HMGA1 and Its Target Slug. Circulation. 2016;133:1783-94.

24. Stenmark KR, Frid M and Perros F. Endothelial-to-Mesenchymal Transition: An Evolving Paradigm and a Promising Therapeutic Target in PAH. Circulation. 2016;133:1734-7.

25. Machado RD, Eickelberg O, Elliott CG, Geraci MW, Hanaoka M, Loyd JE, Newman JH, Phillips JA, 3rd, Soubrier F, Trembath RC and Chung WK. Genetics and genomics of pulmonary arterial hypertension. Journal of the American College of Cardiology. 2009;54:S32-42.

26. Soubrier F, Chung WK, Machado R, Grunig E, Aldred M, Geraci M, Loyd JE, Elliott CG, Trembath RC, Newman JH and Humbert M. Genetics and genomics of pulmonary arterial hypertension. J Am Coll Cardiol. 2013;62:D13-21.

27. van de Veerdonk MC, Kind T, Marcus JT, Mauritz GJ, Heymans MW, Bogaard HJ, Boonstra A, Marques KM, Westerhof N and Vonk-Noordegraaf A. Progressive right ventricular dysfunction in patients with pulmonary arterial hypertension responding to therapy. J Am Coll Cardiol. 2011;58:2511-9.

28. Voelkel NF, Cool C, Lee SD, Wright L, Geraci MW and Tuder RM. Primary pulmonary hypertension between inflammation and cancer. Chest. 1998;114:225S-230S.

29. Rai PR, Cool CD, King JA, Stevens T, Burns N, Winn RA, Kasper M and Voelkel NF. The cancer paradigm of severe pulmonary arterial hypertension. Am J Respir Crit Care Med. 2008;178:558-64.

30. Pullamsetti SS, Savai R, Seeger W, Goncharova EA. Translational Advances in the Field of Pulmonary Hypertension. From Cancer Biology to New Pulmonary Arterial Hypertension Therapeutics. Targeting Cell Growth and Proliferation Signaling Hubs. Am J Respir Crit Care Med. 2017 Feb 15;195(4):425-437.

31. Ghofrani HA, Seeger W and Grimminger F. Imatinib for the treatment of pulmonary arterial hypertension. N Engl J Med. 2005;353:1412-3.

32. Ghofrani HA, Morrell NW, Hoeper MM, Olschewski H, Peacock AJ, Barst RJ, Shapiro S, Golpon H, Toshner M, Grimminger F and Pascoe S. Imatinib in pulmonary arterial hypertension patients with inadequate response to established therapy. Am J Respir Crit Care Med. 2010;182:1171-7.

33. Humbert M. Impression, sunset. Circulation. 2013;127:1098-100.

34. Tuder RM, Groves B, Badesch DB and Voelkel NF. Exuberant endothelial cell growth and elements of inflammation are present in plexiform lesions of pulmonary hypertension. The American journal of pathology. 1994;144:275-85.

35. Rabinovitch M, Guignabert C, Humbert M and Nicolls MR. Inflammation and immunity in the pathogenesis of pulmonary arterial hypertension. Circulation research. 2014;115:165-75.

36. Perros F, Dorfmuller P, Montani D, Hammad H, Waelput W, Girerd B, Raymond N, Mercier O, Mussot S, Cohen-Kaminsky S, Humbert M and Lambrecht BN. Pulmonary lymphoid neogenesis in idiopathic pulmonary arterial hypertension. Am J Respir Crit Care Med. 2012;185:311-21.

37. Paulin R and Michelakis ED. The metabolic theory of pulmonary arterial hypertension. Circulation research. 2014;115:148-64.

38. Sutendra G and Michelakis ED. The metabolic basis of pulmonary arterial hypertension. Cell Metab. 2014;19:558-73.

39. Sutendra G and Michelakis ED. Pulmonary arterial hypertension: challenges in translational research and a vision for change. Science translational medicine. 2013;5:208sr5.

40. Ryan JJ and Archer SL. Emerging concepts in the molecular basis of pulmonary arterial hypertension: part I: metabolic plasticity and mitochondrial dynamics in the pulmonary circulation and right ventricle in pulmonary arterial hypertension. Circulation. 2015;131:1691-702.

41. Cottrill KA and Chan SY. Metabolic dysfunction in pulmonary hypertension: the expanding relevance of the Warburg effect. Eur J Clin Invest. 2013;43:855-65.

42. Archer SL, Fang YH, Ryan JJ and Piao L. Metabolism and bioenergetics in the right ventricle and pulmonary vasculature in pulmonary hypertension. Pulmonary circulation. 2013;3:144-52.

43. Zhao Y, Peng J, Lu C, Hsin M, Mura M, Wu L, Chu L, Zamel R, Machuca T, Waddell T, Liu M, Keshavjee S, Granton J and de Perrot M. Metabolomic heterogeneity of pulmonary arterial hypertension. PLoS One. 2014;9:e88727.

44. Zhao YD, Chu L, Lin K, Granton E, Yin L, Peng J, Hsin M, Wu L, Yu A, Waddell T, Keshavjee S, Granton J and de Perrot M. A Biochemical Approach to Understand the Pathogenesis of Advanced Pulmonary Arterial Hypertension: Metabolomic Profiles of Arginine, Sphingosine-1-Phosphate, and Heme of Human Lung. PLoS One. 2015;10:e0134958.

45. Piao L, Sidhu VK, Fang YH, Ryan JJ, Parikh KS, Hong Z, Toth PT, Morrow E, Kutty S, Lopaschuk GD and Archer SL. FOXO1-mediated upregulation of pyruvate dehydrogenase kinase-4 (PDK4) decreases glucose oxidation and impairs right ventricular function in pulmonary hypertension: therapeutic benefits of dichloroacetate. Journal of molecular medicine. 2013;91:333-46.

46. Guignabert C, Tu L, Izikki M, Dewachter L, Zadigue P, Humbert M, Adnot S, Fadel E and Eddahibi S. Dichloroacetate treatment partially regresses established pulmonary hypertension in mice with SM22alpha-targeted overexpression of the serotonin transporter. FASEB J. 2009;23:4135-47.

47. McMurtry MS, Bonnet S, Wu X, Dyck JR, Haromy A, Hashimoto K and Michelakis ED. Dichloroacetate prevents and reverses pulmonary hypertension by inducing pulmonary artery smooth muscle cell apoptosis. Circulation research. 2004;95:830-40.

48. Michelakis ED, McMurtry MS, Wu XC, Dyck JR, Moudgil R, Hopkins TA, Lopaschuk GD, Puttagunta L, Waite R and Archer SL. Dichloroacetate, a metabolic modulator, prevents and reverses chronic hypoxic pulmonary hypertension in rats: role of increased expression and activity of voltage-gated potassium channels. Circulation. 2002;105:244-50.

49. Ventetuolo CE, Baird GL, Barr RG, Bluemke DA, Fritz JS, Hill NS, Klinger JR, Lima JA, Ouyang P, Palevsky HI, Palmisciano AJ, Krishnan I, Pinder D, Preston IR, Roberts KE and Kawut SM. Higher Estradiol and Lower Dehydroepiandrosterone-Sulfate Levels Are Associated with Pulmonary Arterial Hypertension in Men. American Journal of Respiratory and Critical Care Medicine. 2016;193:1168-75.

50. Pullamsetti S, Kiss L, Ghofrani HA, Voswinckel R, Haredza P, Klepetko W, Aigner C, Fink L, Muyal JP, Weissmann N, Grimminger F, Seeger W and Schermuly RT. Increased levels and reduced catabolism of asymmetric and symmetric dimethylarginines in pulmonary hypertension. FASEB J. 2005;19:1175-7.

51. Nagaya N, Uematsu M, Oya H, Sato N, Sakamaki F, Kyotani S, Ueno K, Nakanishi N, Yamagishi M and Miyatake K. Short-term oral administration of L-arginine improves hemodynamics and exercise capacity in patients with precapillary pulmonary hypertension. Am J Respir Crit Care Med. 2001;163:887-91.

52. Hoet PH and Nemery B. Polyamines in the lung: polyamine uptake and polyamine-linked pathological or toxicological conditions. American Journal of Physiology: Heart and Circulatory Physiology. 2000;278:L417-33.

53. Olson JW, Atkinson JE, Hacker AD, Altiere RJ and Gillespie MN. Suppression of polyamine biosynthesis prevents monocrotaline-induced pulmonary edema and arterial medial thickening. Toxicology and Applied Pharmacology. 1985;81:91-9.

54. Herve P, Launay JM, Scrobohaci ML, Brenot F, Simonneau G, Petitpretz P, Poubeau P, Cerrina J, Duroux P and Drouet L. Increased plasma serotonin in primary pulmonary hypertension. Am J Med. 1995;99:249-54.

55. Jasiewicz M, Moniuszko M, Pawlak D, Knapp M, Rusak M, Kazimierczyk R, Musial WJ, Dabrowska M and Kaminski KA. Activity of the kynurenine pathway and its interplay with immunity in patients with pulmonary arterial hypertension. Heart. 2016;102:230-7.

56. Lewis GD, Ngo D, Hemnes AR, Farrell L, Domos C, Pappagianopoulos PP, Dhakal BP, Souza A, Shi X, Pugh ME, Beloiartsev A, Sinha S, Clish CB and Gerszten RE. Metabolic Profiling of Right Ventricular-Pulmonary Vascular Function Reveals Circulating Biomarkers of Pulmonary Hypertension. Journal of the American College of Cardiology. 2016;67:174-89.

57. Chen J, Tang H, Sysol JR, Moreno-Vinasco L, Shioura KM, Chen T, Gorshkova I, Wang L, Huang LS, Usatyuk PV, Sammani S, Zhou G, Raj JU, Garcia JG, Berdyshev E, Yuan JX, Natarajan V and Machado RF. The sphingosine kinase 1/sphingosine-1-phosphate pathway in pulmonary arterial hypertension. Am J Respir Crit Care Med. 2014;190:1032-43.

58. Rhodes CJ, Ghataorhe P, Wharton J, Rue-Albrecht KC, Hadinnapola C, Watson G, Bleda M, Haimel M, Coghlan G, Corris PA, Howard LS, Kiely DG, Peacock AJ, Pepke-Zaba J, Toshner M, Wort SJ, Gibbs JS, Lawrie A, Graf S, Morrell NW and Wilkins MR. Plasma Metabolomics Implicate Modified Transfer RNAs and Altered Bioenergetics in the Outcome of Pulmonary Arterial Hypertension. Circulation. 2016;135:460-475.

59. Brittain EL, Talati M, Fessel JP, Zhu H, Penner N, Calcutt MW, West JD, Funke M, Lewis GD, Gerszten RE, Hamid R, Pugh ME, Austin ED, Newman JH and Hemnes AR. Fatty Acid Metabolic Defects and Right Ventricular Lipotoxicity in Human Pulmonary Arterial Hypertension. Circulation. 2016;133:1936-44.

60. Deng Z, Morse JH, Slager SL, Cuervo N, Moore KJ, Venetos G, Kalachikov S, Cayanis E, Fischer SG, Barst RJ, Hodge SE and Knowles JA. Familial primary pulmonary hypertension (gene PPH1) is caused by mutations in the bone morphogenetic protein receptor-II gene. Am J Hum Genet. 2000;67:737-44.

61. International PPHC, Lane KB, Machado RD, Pauciulo MW, Thomson JR, Phillips JA, 3rd, Loyd JE, Nichols WC and Trembath RC. Heterozygous germline mutations in BMPR2, encoding a TGF-beta receptor, cause familial primary pulmonary hypertension. Nature genetics. 2000;26:81-4.

62. Newman JH, Trembath RC, Morse JA, Grunig E, Loyd JE, Adnot S, Coccolo F, Ventura C, Phillips JA, 3rd, Knowles JA, Janssen B, Eickelberg O, Eddahibi S, Herve P, Nichols WC and Elliott G. Genetic basis of pulmonary arterial hypertension: current understanding and future directions. J Am Coll Cardiol. 2004;43:33S-39S.

63. Foletta VC, Lim MA, Soosairajah J, Kelly AP, Stanley EG, Shannon M, He W, Das S, Massague J and Bernard O. Direct signaling by the BMP type II receptor via the cytoskeletal regulator LIMK1. J Cell Biol. 2003;162:1089-98.

64. Ma L and Chung WK. The genetic basis of pulmonary arterial hypertension. Human genetics. 2014;133:471-9.

65. Graf S and Morrell NW. Towards a molecular classification of pulmonary arterial hypertension. The European respiratory journal. 2016;48:987-989.

66. Stearman RS, Cornelius AR, Lu X, Conklin DS, Del Rosario MJ, Lowe AM, Elos MT, Fettig LM, Wong RE, Hara N, Cogan JD, Phillips JA, 3rd, Taylor MR, Graham BB, Tuder RM, Loyd JE and Geraci MW. Functional prostacyclin synthase promoter polymorphisms. Impact in pulmonary arterial hypertension. Am J Respir Crit Care Med. 2014;189:1110-20.

67. Wang G, Knight L, Ji R, Lawrence P, Kanaan U, Li L, Das A, Cui B, Zou W, Penny DJ and Fan Y. Early onset severe pulmonary arterial hypertension with 'two-hit' digenic mutations in both BMPR2 and KCNA5 genes. International journal of cardiology. 2014;177:e167-9.

68. Wang H, Li W, Zhang W, Sun K, Song X, Gao S, Zhang C, Hui R and Hu H. Novel promoter and exon mutations of the BMPR2 gene in Chinese patients with pulmonary arterial hypertension. Eur J Hum Genet. 2009;17:1063-9.

69. Aldred MA, Machado RD, James V, Morrell NW and Trembath RC. Characterization of the BMPR2 5'-untranslated region and a novel mutation in pulmonary hypertension. Am J Respir Crit Care Med. 2007;176:819-24.

70. Viales RR, Eichstaedt CA, Ehlken N, Fischer C, Lichtblau M, Grunig E and Hinderhofer K. Mutation in BMPR2 Promoter: A 'Second Hit' for Manifestation of Pulmonary Arterial Hypertension? PLoS One. 2015;10:e0133042.

71. Damico R, Kolb TM, Valera L, Wang L, Housten T, Tedford RJ, Kass DA, Rafaels N, Gao L, Barnes KC, Benza RL, Rand JL, Hamid R, Loyd JE, Robbins IM, Hemnes AR, Chung WK, Austin ED, Drummond MB, Mathai SC and Hassoun PM. Serum endostatin is a genetically determined predictor of survival in pulmonary arterial hypertension. Am J Respir Crit Care Med. 2015;191:208-18.

72. Benza RL, Gomberg-Maitland M, Demarco T, Frost AE, Torbicki A, Langleben D, Pulido T, Correa-Jaque P, Passineau MJ, Wiener HW, Tamari M, Hirota T, Kubo M and Tiwari HK. Endothelin-1 Pathway Polymorphisms and Outcomes in Pulmonary Arterial Hypertension. Am J Respir Crit Care Med. 2015;192:1345-54.

73. Austin ED, West J, Loyd JE and Hemnes AR. Translational Advances in the Field of Pulmonary Hypertension Molecular Medicine of Pulmonary Arterial Hypertension. From Population Genetics to Precision Medicine and Gene Editing. Am J Respir Crit Care Med. 2017;195:23-31.

74. Dawes TJW, de Marvao A, Shi W, Fletcher T, Watson GMJ, Wharton J, Rhodes CJ, Howard LSGE, Gibbs JSR, Rueckert D, Cook SA, Wilkins MR, O'Regan DP. Machine Learning of Three-dimensional Right Ventricular Motion Enables Outcome Prediction in Pulmonary Hypertension: A Cardiac MR Imaging Study. Radiology. 2017 May;283(2):381-390.

75. Voelkel NF, Quaife RA, Leinwand LA, Barst RJ, McGoon MD, Meldrum DR, Dupuis J, Long CS, Rubin LJ, Smart FW, Suzuki YJ, Gladwin M, Denholm EM, Gail DB, National Heart L, Blood Institute Working Group on C and Molecular Mechanisms of Right Heart F. Right ventricular function and failure: report of a National Heart, Lung, and Blood Institute working group on cellular and molecular mechanisms of right heart failure. Circulation. 2006;114:1883-91.

76. Vanderpool RR, Pinsky MR, Naeije R, Deible C, Kosaraju V, Bunner C, Mathier MA, Lacomis J, Champion HC and Simon MA. RV-pulmonary arterial coupling predicts outcome in patients referred for pulmonary hypertension. Heart. 2015;101:37-43.

77. Bogaard HJ, Abe K, Vonk Noordegraaf A and Voelkel NF. The right ventricle under pressure: cellular and molecular mechanisms of right-heart failure in pulmonary hypertension. Chest. 2009;135:794-804.

78. Voeller RK, Aziz A, Maniar HS, Ufere NN, Taggar AK, Bernabe NJ, Jr., Cupps BP and Moon MR. Differential modulation of right ventricular strain and right atrial mechanics in mild vs. severe pressure overload. American journal of physiology Heart and circulatory physiology. 2011;301:H2362-71.

79. Vonk-Noordegraaf A, Haddad F, Chin KM, Forfia PR, Kawut SM, Lumens J, Naeije R, Newman J, Oudiz RJ, Provencher S, Torbicki A, Voelkel NF and Hassoun PM. Right heart adaptation to pulmonary arterial hypertension: physiology and pathobiology. J Am Coll Cardiol. 2013;62:D22-33.

80. Raina A and Humbert M. Risk assessment in pulmonary arterial hypertension. European respiratory review : an official journal of the European Respiratory Society. 2016;25:390-398.

81. Sitbon O, Humbert M, Nunes H, Parent F, Garcia G, Herve P, Rainisio M and Simonneau G. Long-term intravenous epoprostenol infusion in primary pulmonary hypertension: prognostic factors and survival. J Am Coll Cardiol. 2002;40:780-8.

82. McLaughlin VV, Sitbon O, Badesch DB, Barst RJ, Black C, Galie N, Rainisio M, Simonneau G and Rubin LJ. Survival with first-line bosentan in patients with primary pulmonary hypertension. The European respiratory journal. 2005;25:244-9.

83. Nickel N, Golpon H, Greer M, Knudsen L, Olsson K, Westerkamp V, Welte T and Hoeper MM. The prognostic impact of follow-up assessments in patients with idiopathic pulmonary arterial hypertension. The European respiratory journal. 2012;39:589-96.

84. Rich JD, Thenappan T, Freed B, Patel AR, Thisted RA, Childers R and Archer SL. QTc prolongation is associated with impaired right ventricular function and predicts mortality in pulmonary hypertension. International journal of cardiology. 2013;167:669-76.

85. Benza RL, Miller DP, Gomberg-Maitland M, Frantz RP, Foreman AJ, Coffey CS, Frost A, Barst RJ, Badesch DB, Elliott CG, Liou TG and McGoon MD. Predicting survival in pulmonary arterial hypertension: insights from the Registry to Evaluate Early and Long-Term Pulmonary Arterial Hypertension Disease Management (REVEAL). Circulation. 2010;122:164-72.

86. Barst RJ, Chung L, Zamanian RT, Turner M and McGoon MD. Functional class improvement and 3-year survival outcomes in patients with pulmonary arterial hypertension in the REVEAL Registry. Chest. 2013;144:160-8.

87. Savarese G, Paolillo S, Costanzo P, D'Amore C, Cecere M, Losco T, Musella F, Gargiulo P, Marciano C and Perrone-Filardi P. Do changes of 6-minute walk distance predict clinical events in patients with pulmonary arterial hypertension? A meta-analysis of 22 randomized trials. J Am Coll Cardiol. 2012;60:1192-201.

88. Courand PY, Pina Jomir G, Khouatra C, Scheiber C, Turquier S, Glerant JC, Mastroianni B, Gentil B, Blanchet-Legens AS, Dib A, Derumeaux G, Humbert M, Mornex JF, Cordier JF and Cottin V. Prognostic value of right ventricular ejection fraction in pulmonary arterial hypertension. The European respiratory journal. 2015;45:139-49.

89. Raymond RJ, Hinderliter AL, Willis PW, Ralph D, Caldwell EJ, Williams W, Ettinger NA, Hill NS, Summer WR, de Boisblanc B, Schwartz T, Koch G, Clayton LM, Jobsis MM, Crow JW and Long W. Echocardiographic predictors of adverse outcomes in primary pulmonary hypertension. J Am Coll Cardiol. 2002;39:1214-9.

90. Badagliacca R, Poscia R, Pezzuto B, Papa S, Pesce F, Manzi G, Giannetta E, Raineri C, Schina M, Sciomer S, Parola D, Francone M, Carbone I, Fedele F and Vizza CD. Right ventricular concentric hypertrophy and clinical worsening in idiopathic pulmonary arterial hypertension. J Heart Lung Transplant. 2016;35:1321-1329.

91. Swift AJ, Rajaram S, Campbell MJ, Hurdman J, Thomas S, Capener D, Elliot C, Condliffe R, Wild JM and Kiely DG. Prognostic value of cardiovascular magnetic resonance imaging measurements corrected for age and sex in idiopathic pulmonary arterial hypertension. Circ Cardiovasc Imaging. 2014;7:100-6.

92. Fritz JS, Blair C, Oudiz RJ, Dufton C, Olschewski H, Despain D, Gillies H and Kawut SM. Baseline and follow-up 6-min walk distance and brain natriuretic peptide predict 2-year mortality in pulmonary arterial hypertension. Chest. 2013;143:315-23.

93. Nagaya N, Nishikimi T, Uematsu M, Satoh T, Kyotani S, Sakamaki F, Kakishita M, Fukushima K, Okano Y, Nakanishi N, Miyatake K and Kangawa K. Plasma brain natriuretic peptide as a prognostic indicator in patients with primary pulmonary hypertension. Circulation. 2000;102:865-70.

94. Fijalkowska A, Kurzyna M, Torbicki A, Szewczyk G, Florczyk M, Pruszczyk P and Szturmowicz M. Serum N-terminal brain natriuretic peptide as a prognostic parameter in patients with pulmonary hypertension. Chest. 2006;129:1313-21.

95. Leuchte HH, Holzapfel M, Baumgartner RA, Neurohr C, Vogeser M and Behr J. Characterization of brain natriuretic peptide in long-term follow-up of pulmonary arterial hypertension. Chest. 2005;128:2368-2374.

96. Shah SJ, Thenappan T, Rich S, Tian L, Archer SL and Gomberg-Maitland M. Association of serum creatinine with abnormal hemodynamics and mortality in pulmonary arterial hypertension. Circulation. 2008;117:2475-83.

97. Quarck R, Nawrot T, Meyns B and Delcroix M. C-reactive protein: a new predictor of adverse outcome in pulmonary arterial hypertension. J Am Coll Cardiol. 2009;53:1211-8.

98. Kumpers P, Nickel N, Lukasz A, Golpon H, Westerkamp V, Olsson KM, Jonigk D, Maegel L, Bockmeyer CL, David S and Hoeper MM. Circulating angiopoietins in idiopathic pulmonary arterial hypertension. European heart journal. 2010;31:2291-300.

99. Takeda Y, Takeda Y, Tomimoto S, Tani T, Narita H and Kimura G. Bilirubin as a prognostic marker in patients with pulmonary arterial hypertension. BMC Pulm Med. 2010;10:22.

100. Rhodes CJ, Wharton J, Howard LS, Gibbs JS and Wilkins MR. Red cell distribution width outperforms other potential circulating biomarkers in predicting survival in idiopathic pulmonary arterial hypertension. Heart. 2011;97:1054-60.

101. Cracowski JL. Towards prognostic biomarkers in pulmonary arterial hypertension. The European respiratory journal. 2012;39:799-801.

102. Rhodes CJ, Wharton J, Boon RA, Roexe T, Tsang H, Wojciak-Stothard B, Chakrabarti A, Howard LS, Gibbs JS, Lawrie A, Condliffe R, Elliot CA, Kiely DG, Huson L, Ghofrani HA, Tiede H, Schermuly R, Zeiher AM, Dimmeler S and Wilkins MR. Reduced microRNA-150 is associated with poor survival in pulmonary arterial hypertension. Am J Respir Crit Care Med. 2013;187:294-302.

103. Fenster BE, Lasalvia L, Schroeder JD, Smyser J, Silveira LJ, Buckner JK and Brown KK. Cystatin C: a potential biomarker for pulmonary arterial hypertension. Respirology. 2014;19:583-9.

104. Heresi GA, Aytekin M, Hammel JP, Wang S, Chatterjee S and Dweik RA. Plasma interleukin-6 adds prognostic information in pulmonary arterial hypertension. The European respiratory journal. 2014;43:912-4.

105. Zheng YG, Yang T, He JG, Chen G, Liu ZH, Xiong CM, Gu Q, Ni XH and Zhao ZH. Plasma soluble ST2 levels correlate with disease severity and predict clinical worsening in patients with pulmonary arterial hypertension. Clinical cardiology. 2014;37:365-70.

106. Pezzuto B, Badagliacca R, Poscia R, Ghio S, D'Alto M, Vitulo P, Mule M, Albera C, Volterrani M, Fedele F and Vizza CD. Circulating biomarkers in pulmonary arterial hypertension: update and future direction. J Heart Lung Transplant. 2015;34:282-305.

107. Bujak R, Mateo J, Blanco I, Izquierdo-Garcia JL, Dudzik D, Markuszewski MJ, Peinado VI, Laclaustra M, Barbera JA, Barbas C and Ruiz-Cabello J. New Biochemical Insights into the Mechanisms of Pulmonary Arterial Hypertension in Humans. PLoS One. 2016;11:e0160505.

108. Sitbon O, Humbert M, Jais X, Ioos V, Hamid AM, Provencher S, Garcia G, Parent F, Herve P and Simonneau G. Long-term response to calcium channel blockers in idiopathic pulmonary arterial hypertension. Circulation. 2005;111:3105-11.

109. McLaughlin VV, Shah SJ, Souza R and Humbert M. Management of pulmonary arterial hypertension. J Am Coll Cardiol. 2015;65:1976-97.

110. Humbert M, Sitbon O and Simonneau G. Treatment of pulmonary arterial hypertension. N Engl J Med. 2004;351:1425-36.

111. Ghofrani HA, Galie N, Grimminger F, Grunig E, Humbert M, Jing ZC, Keogh AM, Langleben D, Kilama MO, Fritsch A, Neuser D, Rubin LJ and Group P-S. Riociguat for the treatment of pulmonary arterial hypertension. N Engl J Med. 2013;369:330-40.

112. Galie N, Barbera JA, Frost AE, Ghofrani HA, Hoeper MM, McLaughlin VV, Peacock AJ, Simonneau G, Vachiery JL, Grunig E, Oudiz RJ, Vonk-Noordegraaf A, White RJ, Blair C, Gillies H, Miller KL, Harris JH, Langley J, Rubin LJ and Investigators A. Initial Use of Ambrisentan plus Tadalafil in Pulmonary Arterial Hypertension. N Engl J Med. 2015;373:834-44.

113. Sitbon O, Jais X, Savale L, Cottin V, Bergot E, Macari EA, Bouvaist H, Dauphin C, Picard F, Bulifon S, Montani D, Humbert M and Simonneau G. Upfront triple combination therapy in pulmonary arterial hypertension: a pilot study. The European respiratory journal. 2014;43:1691-7.

114. Lythgoe MP, Rhodes CJ, Ghataorhe P, Attard M, Wharton J and Wilkins MR. Why drugs fail in clinical trials in pulmonary arterial hypertension, and strategies to succeed in the future. Pharmacol Ther. 2016;164:195-203.

115. Bonnet S, Provencher S, Guignabert C, Perros F, Boucherat O, Schermuly RT, Hassoun PM, Rabinovitch M, Nicolls MR, Humbert M. Translating Research into Improved Patient Care in Pulmonary Arterial Hypertension. Am J Respir Crit Care Med. 2017 Mar 1;195(5):583-595.

116. Humbert M, Lau EM, Montani D, Jais X, Sitbon O and Simonneau G. Advances in therapeutic interventions for patients with pulmonary arterial hypertension. Circulation. 2014;130:2189-208.

117. Simonneau G, Hoeper MM, McLaughlin V, Rubin L and Galie N. Future perspectives in pulmonary arterial hypertension. European respiratory review : an official journal of the European Respiratory Society. 2016;25:381-389.

118. Thompson AA and Lawrie A. Targeting Vascular Remodeling to Treat Pulmonary Arterial Hypertension. Trends Mol Med. 2017;23:31-45.

119. Chun HJ, Bonnet S and Chan SY. Translational Advances in the Field of Pulmonary Hypertension. Translating MicroRNA Biology in Pulmonary Hypertension. It Will Take More Than "miR" Words. Am J Respir Crit Care Med. 2017;195:167-178.

120. Chambliss AB and Chan DW. Precision medicine: from pharmacogenomics to pharmacoproteomics. Clin Proteomics. 2016;13:25.

121. Brittain EL and Chan SY. Integration of complex data sources to provide biologic insight into pulmonary vascular disease (2015 Grover Conference Series). Pulmonary circulation. 2016;6:251-60.

122. Zhang M, Schmitt-Ulms G, Sato C, Xi Z, Zhang Y, Zhou Y, St George-Hyslop P and Rogaeva E. Drug Repositioning for Alzheimer's Disease Based on Systematic 'omics' Data Mining. PLoS One. 2016;11:e0168812.

123. Lamb J, Crawford ED, Peck D, Modell JW, Blat IC, Wrobel MJ, Lerner J, Brunet JP, Subramanian A, Ross KN, Reich M, Hieronymus H, Wei G, Armstrong SA, Haggarty SJ, Clemons PA, Wei R, Carr SA, Lander ES and Golub TR. The Connectivity Map: using gene-expression signatures to connect small molecules, genes, and disease. Science. 2006;313:1929-35.

124. Wanichthanarak K, Fahrmann JF and Grapov D. Genomic, Proteomic, and Metabolomic Data Integration Strategies. Biomark Insights. 2015;10:1-6.

125. Cambiaghi A, Ferrario M, Masseroli M. Analysis of metabolomic data: tools, current strategies and future challenges for omics data integration. Brief Bioinform. 2016 Apr 12. pii: bbw031

126. Lusis AJ and Weiss JN. Cardiovascular networks: systems-based approaches to cardiovascular disease. Circulation. 2010;121:157-70.

127. Barabasi AL, Gulbahce N and Loscalzo J. Network medicine: a network-based approach to human disease. Nature reviews Genetics. 2011;12:56-68.

128. Vidal M, Cusick ME and Barabasi AL. Interactome networks and human disease. Cell. 2011;144:986-98.

129. Menche J, Sharma A, Kitsak M, Ghiassian SD, Vidal M, Loscalzo J and Barabasi AL. Disease networks. Uncovering disease-disease relationships through the incomplete interactome. Science. 2015;347:1257601.

130. Parikh VN, Jin RC, Rabello S, Gulbahce N, White K, Hale A, Cottrill KA, Shaik RS, Waxman AB, Zhang YY, Maron BA, Hartner JC, Fujiwara Y, Orkin SH, Haley KJ, Barabasi AL, Loscalzo J and Chan SY. MicroRNA-21 integrates pathogenic signaling to control pulmonary hypertension: results of a network bioinformatics approach. Circulation. 2012;125:1520-32.