today’s and tomorrow’s imaging and circulating biomarkers ... · 2) pulmonary hypertension with...

REVIEW

Today’s and tomorrow’s imaging and circulating biomarkersfor pulmonary arterial hypertension

Marjorie Barrier • Jolyane Meloche • Maria Helena Jacob •

Audrey Courboulin • Steeve Provencher • Sebastien Bonnet

Received: 17 October 2011 / Revised: 18 February 2012 / Accepted: 20 February 2012 / Published online: 25 March 2012

� Springer Basel AG 2012

Abstract The pathobiology of pulmonary arterial

hypertension (PAH) involves a remodeling process in distal

pulmonary arteries, as well as vasoconstriction and in situ

thrombosis, leading to an increase in pulmonary vascular

resistance, right heart failure and death. Its etiology may be

idiopathic, but PAH is also frequently associated with

underlying conditions such as connective tissue diseases.

During the past decade, more than welcome novel therapies

have been developed and are in development, including

those increasingly targeting the remodeling process. These

therapeutic options modestly increase the patients’ long-

term survival, now approaching 60% at 5 years. However,

non-invasive tools for confirming PAH diagnosis, and

assessing disease severity and response to therapy, are

tragically lacking and would help to select the best treat-

ment. After exclusion of other causes of pulmonary

hypertension, a final diagnosis still relies on right heart

catheterization, an invasive technique which cannot be

repeated as often as an optimal follow-up might require.

Similarly, other techniques and biomarkers used for

assessing disease severity and response to treatment gen-

erally lack specificity and have significant limitations. In

this review, imaging as well as current and future circu-

lating biomarkers for diagnosis, prognosis, and follow-up

are discussed.

Keywords Pulmonary arterial hypertension �Biomarkers � Imaging � Circulating biomarkers

Abbreviations

5-HT Serotonin or 5-hydroxytryptamine

BMP Bone morphogenetic protein

BMPRII BMP receptor 2

BNP Brain natriuretic peptide

cGMP Cyclic guanosine 3050-monophosphate

CT Computed tomography

CTEPH Chronic thromboembolic pulmonary

hypertension

DCE Delayed contrast enhancement

Echo Echocardiography

EGF Epidermal growth factor

eNOS Endothelial nitric oxide synthase

ET-1/3 Endothelin 1 or 3

FC Functional class

HDL-C High-density lipoprotein cholesterol

IL-6 Interleukin 6

iPAH Idiopathic PAH

LIGHT Lymphotoxin-like inducible protein

LV Left ventricle

miR-X MicroRNA-X

miRNA Micro RNA

MP Microparticles

MRI Magnetic resonance imaging

NFAT Nuclear factor of activated T cells

NO Nitric oxide

NT-proBNP N-terminal pro-brain natriuretic peptide

OPN Osteopontin

PA Pulmonary arteries

PAAT Pulmonary artery acceleration time

PAH Pulmonary arterial hypertension

M. Barrier � J. Meloche � M. H. Jacob � A. Courboulin �S. Provencher � S. Bonnet (&)

Pulmonary Hypertension Research Group, Centre de Recherche

de l’Institut Universitaire de Cardiologie et de Pneumologie de

Quebec, 2725 Chemin Ste-Foy, Quebec, QC G1V 4G5, Canada

e-mail: [email protected]

Cell. Mol. Life Sci. (2012) 69:2805–2831

DOI 10.1007/s00018-012-0950-4 Cellular and Molecular Life Sciences

123

PAP Pulmonary arterial pressure

PASMC Pulmonary artery smooth muscle cell

PDE5 Phosphodiesterase 5

PDGF Platelet-derived growth factor

PECAM Platelet endithelial cell adhesion molecule

PET Position emission tomography

PH Pulmonary hypertension

Pim1 Proviral integration site for Moloney murine

leukemia 1

PPAR Peroxisome proliferator activated receptor

(gamma c or alpha a)

Q Pefusion

RDW Red blood cell distribution width

RHC Right heart catheterization

ROS Reactive oxygen species

RV Right ventricle

RVH Right ventricle hypertrophy

SPECT Single photon emission computed

tomography

SOD Superoxyde dismutase

STAT3 Signal transducer and activator of

transcription 3

TAPSE Tricuspid annular plane systolic excursion

TGFb Tranforming growth factor beta

TXA2 Thromboxane A2

V Ventilation

vWF Plasma von Willebrand factor

WHO World Health Organization

Introduction

Pulmonary hypertension (PH) includes several pathologies.

A new classification of PH designated five distinct cate-

gories differing in their pathophysiology, clinical

presentation, diagnostic findings, and response to treatment

(Table 1) [1]. Amongst these, pulmonary arterial hyper-

tension (PAH) is characterized by pulmonary vascular

remodeling featured by vasoconstriction, remodeling of the

small pulmonary artery (PA) wall, and in situ thrombosis

[2]. PAH is clinically characterized by progressively

increasing pulmonary vascular resistance eventually lead-

ing to right ventricle (RV) hypertrophy (RVH), failure and

death [3, 4]. PAH may be idiopathic or associated with

various conditions (Table 1) [5].

Biomarkers are obviously an essential missing element

for diagnosing PAH [1, 6]. In fact, the average delay before

diagnosing PAH patients is 2.8 years, a period during

which the disease often worsens [7]. The long current delay

is explained by the lack of specificity of PAH symptoms. In

fact, dyspnea is the cardinal symptom of PAH and is

generally attributed to other pulmonary diseases such as

asthma and chronic obstructive pulmonary disease, or to

depression and anxiety disorders, before being associated with

PAH. Moreover, the prevalence of PAH is estimated at 60 per

million, which makes it rare and even more difficult to diag-

nose, since some physicians are not familiar with PAH [8].

Thus, new diagnostic tools should ideally reduce this delay in

order to treat this pathology at its earliest stages. Indeed, PAH

is diagnosed by an exclusion principle to strike out the other

PH groups. Among the PAH group (group 1), iPAH is a

diagnostic of exclusion since no known cause is related to the

disease, as it is idiopathic (Fig. 1).

Fortunately, major improvements have been made in the

treatment of PAH in the past decade. Despite these novel

therapies, most patients still display persistent exercise

intolerance, and the long-term prognosis remains poor [4,

9–12]. A goal-oriented therapy has been proposed to

improve long-term outcomes of PAH patients [13].

Table 1 Classification of pulmonary hypertension (PH)

1) Pulmonary arterial hypertension (PAH)

Idiopathic (iPAH)

Heritable

Related with collagen vascular disease (e.g., systemic sclerosis),

congenital left-to-right shunt, HIV infection, use of drugs or toxins,

portal hypertension or other causes (e.g., scleroderma)

Pulmonary hypertension of the newborn

Pulmonary veino-occlusive disease

Pulmonary capillary hemangiomatosis

2) Pulmonary hypertension with left-sided heart disease

Systolic dysfunction

Diastolic dysfunction

Valvular disease

3) Pulmonary hypertension associated with lung diseases and/or

hypoxemia

Chronic obstructive pulmonary disease (COPD)

Interstitial lung disease

Sleep-disordered breathing, alveolar hypoventilation

Chronic exposure to high altitude

Developmental lung abnormalities

4) Pulmonary hypertension due to chronic thrombotic and/or embolic

disease

Chronic thromboembolic pulmonary hypertension (CTEPH) in

proximal or distal pulmonary arteries

Embolization of other matter, such as tumor, parasites or foreign

material

5) Pulmonary hypertension with unclear multifactorial mechanisms

Hematologic disorders (i.e. myeloproliferative disorders,

splenectomy)

Systemic disorder (i.e. sarcoidosis)

Metabolic disorder (i.e. glycogen storage disease)

Other causes like compression of pulmonary vessels, chronic renal

failure

HIV Human immunodeficiency virus

2806 M. Barrier et al.

123

However, this proposal has emphasized the lack of a con-

sistent protocol for evaluating the course of the disease.

Indeed, establishing such goals is hampered by the gap in

the evidence-based assessment of PAH treatment efficacy.

Only limited tools are currently available for predicting the

long-term outcome in PAH. An accurate biomarker should

guide the escalation of pharmacotherapy given the risk/

benefit ratio and the timing of referral for lung/heart

transplantation, i.e. the ultimate tool for saving patients

from RV failure. This involves the capacity to evaluate the

improvement or degradation of the patient’s condition. In

such circumstances, referral for life-saving interventions

such as lung transplantation would not be uselessly delayed

in the significant portion of patients who are unresponsive

to new therapies [14].

Current understanding of PAH pathophysiology

Pulmonary arterial hypertension is a vascular disease that is

largely restricted to small PA. Many abnormalities con-

tribute to this syndrome of obstructed, constricted small

PA. This includes abnormalities in blood concentration of a

few growth factors, neurotransmitters and cytokines,

namely increases in platelet-derived growth factor (PDGF),

serotonin, interleukin-6 (IL-6), and endothelin-1 (ET-1).

Nonetheless, these factors may not be reliable biomarkers

since they are increased by stress, inflammation, and other

common assaults on body integrity, and therefore, may not

be specific to PAH. The media in PAH also exhibits an

increased activation of the NFAT (nuclear factor of acti-

vated T cells) transcription factor, leading to increased

Ca2?-dependent PA smooth muscle cell (PASMC) prolif-

eration and decreased mitochondrial-dependent apoptosis

[15]. There is also a metabolic disorder due in part to an

impairment of mitochondrial metabolism known as the

Warburg effect, which is defined as a decrease in glucose

oxidation and an increase in glycolysis [16]. This phe-

nomenon is well described in cancer, and mounting

evidence supports the fact that PAH also displays a War-

burg effect [16]. Finally, the adventitia is infiltrated with

inflammatory cells and exhibits metalloprotease activation

[17]. Despite recent advances in the therapy of PAH,

mortality rates remain high (40% at 5 years) [18]. Over the

last 5 years, many investigators (including our laboratory)

Suspected pulmonary hypertension

Dyspnea on exertion, syncope, other predisposition to PH (family history, sleep apnea, …)

Echocardiography

Normal echocardiography with high clinical suspicion

Elevated right ventricle systolic pressure and RV hypertrophy

Imaging testing•Pulmonary function testing

•Chest radiography•Ventilation-perfusion scan

•CT-scan•Magnetic resonance imaging

Confirmation by right heart catheterization(mPAP1 > 25mmHg)

Pulmonary arterial hypertension

(group 1)

Pulmonary hypertension associated with another disease

(group 2, 3, 4 or 5)

Serologic testing•Complete blood count

•Human immunodeficiencyvirus testing

•Circulating biomarker evaluation (BNP, troponin T, uric acid)

Other•Liver function test•Six-minute walked

distance test•Sleep study may be

considered

No evidence of underlying disease of

pulmonary hypertension

Evidence of underlying disease of pulmonary

hypertension

Confirmation by right heart catheterization(mPAP1 > 25mmHg)

Fig. 1 Pulmonary hypertension

diagnostic algorithm. This

represents the usual algorithm

followed by patients with

suspected pulmonary

hypertension to diagnose the

class of pulmonary hypertension

in order to better adapt the

treatment

Today’s and tomorrow’s imaging and circulating biomarkers 2807

123

have endeavored to understand the mechanisms for

enhanced PASMC proliferation and decreased apoptosis,

which account for distal PA remodeling. Indeed, the lung

peripheral vascular bed is increasingly the target of novel

treatment strategies, which aim at restoring physiological

vascular tone and at reducing the remodeling process [19].

Many pathways are implicated in PAH development since

this pathology is a combination of many factors such as

PASMC deregulation (enhanced proliferation and migra-

tion, resistance to apoptosis), abnormal endothelial cells

(causing endothelial barrier disruption and plexiform

lesions), and inflammation as well as increased elastolytic

activity. Many factors have been shown to be implicated in

PAH pathogenesis. Among others, RhoA/ROCK, BMPRII

(bone morphgenetic protein receptor II) dysfunction,

STAT3 (signal transducer and activator of transcription-3),

and NFATc2 upregulation seem to play a critical role in

PASMC proliferation. The issue is that neither STAT3, an

enhancer of NFAT expression, nor NFAT itself can be

targeted, because of their critical function in a plethora of

physiological processes, such as the immune response.

More recently, our group has demonstrated that NFAT

expression is STAT3-dependent while its activation relies

on the oncogene protein Pim1 (proviral integration site for

Moloney murine leukemia) [20]. Inflammatory factors such

as TGF-b, IL-6, and BMP can stimulate PASMC prolif-

eration [21]. Modification in BMPRII levels can trigger a

large amount of signaling pathways. For example, tran-

scription factor PPARc (peroxisome proliferator activated

receptor gamma) is decreased by BMPRII dysfunction and

Rabinovich et al. remarkably showed that PPARc is

implicated in PASMC proliferation and migration, but also

in endothelial cells dysfunction contributing to PAH

pathobiology [22–24]. MicroRNA (miRNA) are also

implicated in the sustainability of the disease, Courboulin

et al. have indeed identified miR-204 as implicated in PAH.

Other microRNA, such as miR-21, miR-34c, and miR-133,

are implicated in right ventricular failure and, thus, may

also play a role in PAH [25, 26]. These factors, proteins,

and pathways are not yet used as biomarkers nor thera-

peutic targets, but this shows that the past decade brought

knowledge in understanding PAH in a way to potentially

treat it.

In this review, currently available biomarkers will be

presented together with their limitations and more recent

improvements.

Primary tests

The first signs of PAH are observed by physical exami-

nation, including an increase in jugular venous distension,

a holosystolic murmur of tricuspid valve regurgitation and

a loud P2. Hepatomegaly, peripheral edema, ascites, and

cool extremities are eventually observed when RV fail-

ure is more severe. Electrocardiography is usually

performed at this stage, highlighting a right axis devia-

tion, RV hypertrophy, and peaked P-waves. Moreover,

nocturnal hypoxemia occurs in [75% of PAH patients,

independently of the occurrence of apneas or hypopneas

[27]. Isolated alteration in diffusing lung capacity for

carbon monoxide is traditionally seen on pulmonary

function tests, although pulmonary function may be

normal or exhibits mild obstructive/restive disorder in

PAH patients [28, 29]. When cardiopulmonary exercise

testing is performed, PAH patients generally display

altered cardiac response and gas exchange abnormalities

[30]. However, these abnormalities are generally seen

when right heart failure is already present and lack

specificity.

New York Heart Association/World Health

Organization functional class

The New York Heart Association and World Health

Organization (WHO) functional classes (FC) are based on

symptoms of dyspnea with degree of exercise intolerance

[31], and help to classify PAH severity. PAH severity is

most commonly assessed objectively with the use of WHO

functional class combined with exercise capacity and RV

function. Various studies have shown that FC and exercise

capacity as assessed by the cardiopulmonary exercise test

[32–34] or the 6-min walk test [34–37] have good dis-

criminating properties and predict survival in PAH [35].

However, caution must be exerted when using the func-

tional classification as an endpoint since the assignment of

patients to different classes is subject to investigator and

patient bias, even if they have held up in multivariate

analysis [31, 35, 38, 39].

Right heart catheterization (RHC)

Right heart catheterization is the gold standard for the

diagnosis of PH. It is to date the only direct proof of PH.

PH is defined as a mean pulmonary artery pressure (PAP)

at rest [25 mmHg (Fig. 1). The indication for RHC relies

on the estimated systolic PAP on echocardiography, the

presence of additional echocardiographic findings sugges-

tive of PH and the presence of symptoms or risks factors

for PH [40]. Crucial information such as PAP (systolic,

diastolic and mean) values, right arterial and ventricular

pressures, PA wedge pressure, pulmonary and systemic

vascular resistance, cardiac output and cardiac index and

mixed venous oxygen saturation can all be measured by


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catheterization [41]. RHC is essential for therapy selec-

tion since it provides important data on pulmonary

circulation vasoreactivity [41], and provides important

information about disease severity and response to ther-

apy. Nonetheless, the technique is invasive, although the

incidence of complications in experienced centers is small

[42–44].

Imaging

Imaging represents a powerful tool for assessing lung

peripheral vascular bed, as well as heart function and

morphology. Indeed, although PAH patients suffer from a

lung disease, functional capacity and prognosis is inti-

mately related to heart function [45]. Thus, it is not only

critical to be able to observe improvements at the lung

level but also to visualize and analyze RV functions.

Actually, the complex and unique shape and the contractile

pattern of the RV make it more difficult to assess [46].

Different imaging techniques are now available to assess

lungs as well as the RV. The development of new non-

invasive biomarkers would be of great interest at diagnosis,

but also for follow-up.

Echocardiography

Echocardiography is a key screening tool in the diagnostic

algorithm of PAH (Fig. 1). In comparison with invasive

measurements, it has the advantages of being safe, easily

and rapidly performed, portable, widely available and thus

well suited for screening, despite the fact that it is con-

sidered performer-dependent [47]. After PH is diagnosed

by RHC, disease monitoring by echocardiography is done

every 3–6 months.

Baseline measurements

Doppler echo is performed on patients with suspected PAH

to assess several parameters such as heart function and

shape, as well as pulmonary and cardiac hemodynamics.

Actually, echo allows the assessment of right and left

heart chamber sizes, and PAH patients display an enlarged

right atrial and RV size and RVH, instead of a crescent-

shaped RV. Moreover, it is also possible to see pericardial

effusions and diastolic septal shift [48], which have prog-

nostic values for PAH patients [48, 49]. Echo also allows

exclusion of other types of PH, including valvular dys-

function, congestive heart failure and congenital heart

disease.

Furthermore, RVSP can be estimated by tricuspid

regurgitation using Doppler, right arterial pressure and

pulmonary artery acceleration time (PAAT). PAAT is

decreased in PAH patients and known to correlate with

disease severity. mPAP can also be approximated using

Doppler function, with a correlation varying from 0.57 to

0.93 compared to PAP measured by RHC. PAP measured

by echo also allows the calculation of other echo-derived

parameters (e.g., aorta diameter, pulmonary velocity, heart

rate), and the calculation of PVR, which is well correlated

to disease severity [50]. LV eccentricity index has also

been shown to have some predictive value on PAH patients

outcome [8, 48].

Some findings have also highlighted the prognostic

values of the Tei-Index and the tricuspid annular plane

systolic excursion (TAPSE) [51]. The Tei-Index, already

used to evaluate the performance of the myocardium, has

proved useful in the evaluation of ventricular function.

Tricuspid annular displacement has also shown good

results in the prognosis of PH patients [52]. Furthermore,

systolic displacement of the tricuspid annulus towards the

RV apex is referred as TAPSE, correlates with RV ejection

fraction, and powerfully reflects RV function. A decreased

TAPSE portends a poor prognosis in patients with dilated

cardiomyopathy [53–55], and Forfia et al. [56] showed that

there is also a correlation between TAPSE and PAH

prognosis (survival) in PAH patients. The latter authors

have also demonstrated a good correlation between TAPSE

values and mPAP, but an even better one with PVR. Fur-

thermore, TAPSE is highly reproducible, unlike mPAP

estimated by echocardiography. Nonetheless, further stud-

ies have to be performed to confirm these results.

Follow-up values

Echocardiography is also a good method to monitor ther-

apeutic efficiency on RV function as it is a critical element

of PAH prognosis and since it can be visualized on echo.

Indeed, echo is also used to assess the efficiency of treat-

ment as a follow-up technique. PAH deterioration is

accelerated in PAH patients with RV dysfunction [4, 57].

Indeed, this imaging technique assesses parameters such as

ventricular and septal morphologies, and maximal velocity

of tricuspid regurgitation jets. All these parameters are

significantly different between the intravenous epoprost-

enol (a prostacyclin analogous) and control groups [58].

Moreover, in another randomized controlled trial, a sig-

nificant difference in changes in ventricular morphology,

i.e. the minimum diameter of the inferior vena cava and

Doppler measurement (RV ejection time and mitral valve

peak velocity) in the patients taking bosentan (a endothelin

receptor antagonist) was observed [8, 58], indicating that

echo can be used as a follow-up technique.

Echo also represents a useful tool in the early diagnosis

of PH. Indeed, first-degree relatives of PAH patients should

undergo routine echo every 3–5 years to ensure that no


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hereditary PAH is developing, or if symptoms manifest

themselves in order to get a diagnosis as quickly as pos-

sible if they develop PAH [41, 43, 59]. Echo is also useful

in screening other at-risk population including scleroderma

and HIV-positive patients [41, 60, 61].

Limitations and future developments

The main issue with this technique is its poor inter-obser-

ver agreement, and, due to its 2-D assessment, heart shape

based on certain assumptions. Moreover, the ultrasound

window can be limited not only because of high body mass

index (BMI), lung fibrosis, or emphysema but also due to

the sternum position of the patient [52]. Like other imaging

techniques [magnetic resonance imaging (MRI), computed

tomography (CT) scan], echo is constantly improving and

has been shown to be accurate in assessing RV function

[62]. Nonetheless, despite all its disadvantages, echo is

likely to remain the most prominent tool for screening PAH

and for follow-up, at least for the time being. This is

explained by its availability and, despite its lack of preci-

sion, this imaging technique has nonetheless amply proven

its reliability for screening. Furthermore, this imaging tool

undergoes technical improvement.

The RV has a very complicated shape, which is hard to

assess by echo due to 2-D limitations. Some authors

believe that assessing LV compression yields more reliable

results than RVH [52]. The 3-D echocardiography is in

progress, which is of a great interest since it would

decrease the requirements for geometric assumptions and

provide more accurate estimations of LV and RV volumes

and ejection fractions [63, 64]. New improvements are also

expected concerning insights into RV and LV structure,

function and interdependence [8, 65].

Recently, a novel application for Doppler echocardiog-

raphy permits RV strain analysis, a very promising

development [66]. Indeed, in a recent study in 30 PAH

patients, Filusch et al. have shown that RV systolic strain

and strain rate were significantly altered and correlated

with levels of N-terminal pro-brain natriuretic peptide (NT-

proBNP, a serum biomarker) and reduced 6MWD com-

pared to matched control. Moreover, reduced strains also

correlated with both mPAP and PVR in these PAH patients

[51]. Thus, detection of the strains is able to discriminate

between the different WHO FC and predict in which class

patients will fall. Results from RV strain analysis are also

related to invasive pulmonary hemodynamic parameters

such as PVR, mPAP and cardiac outpout, emphasizing its

prognostic value [47, 67].

Unfortunately, some of the parameters discussed above

may sometimes show lack of specificity. This explains why

RHC must be made on patient with normal echo-derived

RVSP but with high suspicion of PAH, to ensure whether

or not this patient suffers PAH. Indeed, RHC remains the

only diagnostic measure for PAH, and is usually used to

confirm the presence of PAH. Nonetheless, echo is a pre-

cious tool in screening patients with PAH-like symptoms,

to at least exclude pulmonary pathologies, if not being a

PAH diagnosis tool itself.

Radionuclide imagery

This type of imaging technique includes single photon

emission computed tomography (SPECT), positron emis-

sion tomography (PET), scintigraphy, and magnetic

resonance spectroscopy imaging [67]. A few decades ago,

before the emergence of CT and MRI, radionuclide

imaging was commonly used [47]. The presence of per-

fusion (Q) abnormalities using perfusion scintigraphy has

already been shown in late PAH. However, in addition to

impairment in perfusion, there is also a concomitant

impairment in ventilation (V) as seen by planar scintigra-

phy [68].

Using SPECT, which is more sensitive than planar

scintigraphy for assessing focal V–Q disturbances, the V/Q

SPECT-derived ratio can be measured and provides topo-

graphic V–Q distribution and an objective quantification of

cross-sectional lung V–Q imbalance with a correlation of

lung morphology on CT. Some groups have been able to

find V impairment resulting in V–Q mismatch in patients

with mild PAH. As indicated by the significant correlation

observed between the standard deviation of the entire lung

V/Q ratios and the mean PAP in these patients, the lung

pathophysiology causing V–Q imbalance may be more

advanced, as shown by persistently elevated PAP in iPAH

[68]. However, the main indication for lung V/Q scan

remains to date the exclusion of chronic thromboembolism

as the cause of PH [69].

Using PET scan, Wong et al. demonstrated that RV

myocardial blood flow is increased in iPAH, whereas high

O2 extraction fraction was associated with poorer NYHA

class and more severe RV failure. This may explain why an

additional demand to the heart may lead to cardiac ische-

mia and RV failure [70].

Another interesting aspect is the Warburg effect since it

is a hallmark of both cancer and PAH [16]. This patho-

logical phenomenon is characterized by an imbalance

between oxidative phosphorylation and glycolysis, pro-

moting glycolysis and lactate production. This also

involves increased glucose uptake compared to non-

pathological tissues. The PET scan is already well developed

and commonly used in cancer to assess the development of

a Warburg effect and response to treatment, by correlating

that effect with tumor severity and aggressiveness. Thus,

this technique may be thought as also being of some

prognostic value in PAH management. Indeed, using PET,


123

Abikhzer et al. [71] have reported that RV, which is usually

invisible in PET, could be imaged in a PAH patient due to

increased glucose uptake. This might represent a future

approach to assess RVH severity. Moreover, Marshboom

et al. have recently demonstrated that this technique may

also be possible as assessed the PAH lungs, as it also

displays Warburg effect [72].

New tools are also emerging, such as radioligand

binding of phosphodiesterase 5 (PDE5) [73, 74] and ET-1

[75], which are currently being evaluated in animal models.

Indeed, these receptors are already targeted in the patho-

physiology of PAH, and the hypothesis that their level is

related to the evolution of patients’ state is under scrutiny.

Thus, it might be interesting to verify the relevance of such

radioligands by quantifying the changes in the expression

of PDE5 and ET-1 as biomarkers in patients. These end-

points will obviously require further studies and will need

to be validated more extensively in PAH animal models

before going through human trials.

Magnetic resonance spectroscopy imaging has much

contributed to the diagnosis and prognosis of cancer

through the study of the metabolic changes occurring in

tumors. Thus, this technique might also bring a contribu-

tion to the field of PAH. Moreover, this imaging tool

supplements the use of MRI in cancer in order to facilitate

the diagnosis and to decide upon a proper therapeutic

strategy. Hence, it might well be possible to apply such a

combination of techniques to PAH [76].

CT scan

The gating system for the lung and heart (based on the elec-

trocardiogram gating system) has been a major improvement

in CT, allowing to overcome artifacts due to respiration and

heartbeat. This allowed the functional assessment of the heart

in addition to lung parameters. Indeed, it is believed that RV

remodeling follows PA remodeling. Thus, it might be of

crucial interest to assess PA remodeling before the effect can

be visualized in the right heart as a result of disease progres-

sion. Moreover, assessing lung remodeling would allow

assessing the response to treatment in distal PA where the

pathophysiological process actually takes place.


Some investigators have studied the main PA diameter in

relationship to mean PAP. Unfortunately, its correlation

with mean PAP varies from weak to strong even when

corrected for the size of the aorta, the thoracic vertebrae or

the arterio-bronchial segment in more than three lobes [77,

78], and for the BMI [79]. Moreover, a progressive dila-

tation of the main PA overtime is frequently discrepant

with PAH severity [80].

More importantly, images obtained by CT may allow

specialists to visualize characteristics that help in the dif-

ferential diagnosis of PAH. For example, the combination

of mosaic lung attenuations, a marked variation in the size

of segmental vessels, peripheral parenchymal opacities as

well as dilatation of bronchial and non-bronchial systemic

arteries is common in chronic thromboembolic pulmonary

hypertension (CTEPH) (73%) but is rare in PAH (14%),

which can provide a differential diagnosis between these

two pathologies [77]. Similarly, the CT scan allows the

exclusion of severe parenchymal abnormalities responsible

for PH (e.g., pulmonary fibrosis), and can identify other

possible causes of PH (congenital malformations, coronary

heart disease or cardiac dysfunction) [81]. Finally, it may

document pericardial effusions, which is directly related to

PAH severity [77].

More recently, data from electrocardiographically-gated

multidirectional CT studies have shown that functional

parameters such as right PA dispensability, systolic–dia-

stolic RV outflow tract dimensions, and diastolic wall

thickness are measurable with good inter-observer agree-

ment, and can reliably be used as criteria for PAH

diagnosis [77]. In a recent study, Revel et al. [82] found

that the most reliable parameter among all electro-

cardiographically-gated CT parameters evaluated for

identifying PAH patients is right PA wall distensibility.

This measure also correlates with mean PAP [71]. The

right ventricle/left ventricle ratio, using a gated or non-

gated multi-directed CT reflects RV function if acute pul-

monary embolism. Whether these results can be extended

to PAH patients remains unknown [83].

Devaraj et al. [78] have tested a composite index using

the CT-derived ratio of the diameter of the ascending aorta

and echo-derived RV systolic pressure. The combination of

these two parameters from two different imaging tech-

niques showed a relationship with mean PAP and was a

better predictor of PH than either measurement alone. That

index has been found to increase specificity to 100% with

respect to RHC-derived diagnosis compared to the criterion

of main PA diameter alone [84]. Indeed, a combination of

the (CT-derived main PA diameter)/(ascending aorta) ratio

and echo-derived RV systolic pressure has been found to

better correlate with RHC-derived mean PAP than either

single measurement [78]. A simultaneous use of parame-

ters derived from CT and echo also represents a more

informative combination since CT provides anatomical

data whereas echo identifies their functional implications.

Limitations and future developments

A major limitation of CT-scan is X-ray exposure, which is

problematic when multiple follow-up measures are neces-

sary. Nevertheless, major improvements are currently


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made to limit X-ray levels and the exposure time required

for a given scan quality [85]. Moreover, the contrast agents

used may exhibit significant nephrotoxicity and be aller-

genic in some patients (e.g., iodine). Claustrophobia is a

more mundane limitation that can be taken into account.

New investigations based on technological improve-

ments are constantly being undertaken to circumvent

limitations related to radionuclide imaging, the technique

replaced by CT-scan, but also related to CT-scan itself.

Dual-energy CT-perfusion imaging is a new technology

that decreases X-ray exposure with results comparable to

perfusion scintigraphy. That technique can provide CT

pulmonary angiograms, high-resolution morphologic ima-

ges, and spatially matched perfusion images in the very

same scan, with the same radiation dose that is sustained in

a classic CT pulmonary angiogram. Nevertheless, a few

technical issues remain to be addressed [86, 87]. Another

novel technique is area-detector CT, which will probably

soon be developed in the lung, although radiation exposure

remains significant. The CT-scan is important as it allows

the exclusion of underlying pathologies related to PH.

Furthermore, new types of image acquisition allow the

collecting of increasing amounts of data in a single scan-

ning session.

MRI

MRI has represented a major advance in the assessment of

lung and cardiac anatomy and function. Compared to echo,

MRI provides a higher spatial resolution and exhibits better

inter-observer reproducibility [88], and one of its major

advantages is that planes can be moved, and due to the 3-D

view, no approximation is required. Thus, cardiac MRI is

now considered as the gold standard for the detailed study

of the RV and is currently used as an endpoint in the

multinational European Union-funded Framework 6

EURO-MR project for PH, which will provide an impor-

tant opportunity to more definitely establish its utilization

as an endpoint [89]. Moreover, recent improvements of

hardware and software currently enables semi-automated

myocardial blood border definition, reducing intra- and

inter-observer variability in ventricular volume measure-

ments and decreasing the time spent on analysis [8, 52, 90–

95]. Finally, technical improvements of cine acquisition

have enabled the analysis of dynamic visualization of

several heartbeats (as cine images) with a significant

decrease in breath-holding [46], which is frequently prob-

lematic in PAH patients who usually complain of dyspnea.


Cardiac MRI is most commonly used to assess end-systolic

and end-diastolic RV and LV volumes and the resulting

RV ejection fraction, stroke volume and cardiac output.

Cardiac output can also be assessed by cine phase contrast

methods measuring volumetric flow in the main PA. Not

surprisingly, the RV end-diastolic and end-systolic vol-

umes have been shown to be significantly increased,

whereas stroke volume and cardiac output are decreased in

PAH compared to controls. Moreover, the RV ejection

fraction has been described as being impaired by *50% in

PAH patients [52]. MRI also allows the assessment of RV

hypertrophy, including (1) RV mass, which is increased

two- to threefold in PAH [52], (2) the Fulton ratio (RV

mass/LV mass with septum), which is increased by 80% in

PH patients, and (3) end-systolic RV wall thickness. As RV

failure progresses, LV end-diastolic volume and peak LV

filling rate are impaired in PAH patients and may be assessed

with cardiac MRI [52, 96]. In order to assess cardiac function

under stress conditions, some authors have proposed cardiac

MRI using dobutamine, a b1-adrenergic agonist [97], or

during exercise using a MR-compatible ergometer [98].

These experiments documented the incapacity of PAH

patients to increase stroke volume from rest to exercise.

Delayed contrast enhancement (DCE) has been found to

be frequent in PAH patients, especially when associated

with connective tissue disease [99]. DCE is mainly

observed at the RV intersection points and in intraven-

tricular septum area. Although DCE may be associated

with myocardial ischemia, it is more consistent with

myocardial fibrosis. The extent of DCE in the myocardium

has been shown to be inversely related to measures of RV

functions [100]. Finally, PAH patients have been shown to

display an interventricular asynchrony caused by a longer

RV systolic contraction using tagged MRI [46]. Cardiac

MRI is also of value to exclude other causes of PH such as

congenital heart disease of LV impairment (e.g., diastolic

dysfunction, constrictive pericarditis).

MRI can also be used to assess the pulmonary circula-

tion. MRI-measured proximal PA pulsatility, a marker of

PA stiffness predicts mortality in PAH patients [101].

However, the correlation between PA distensibility and

RHC-derived mean PAP remains controversial [101, 102].

Furthermore, 2-D MR angiograms have been reported to

differentiate CTEPH from PAH with a sensitivity of 92%

compared to a V/Q scan [103]. Similarly, Nikolaou et al.

[104] have been able to differentiate CTEPH from PAH

with an impressive accuracy of 90%, using contrast

enhanced perfusion MRI and MR angiograms with parallel

imaging techniques [104]. Moreover, PAH patients have

significantly decreased pulmonary blood flow and pro-

longed mean time transit in the whole lung, as documented

by 3-D dynamic contrast-enhanced perfusion MRI [104].

Nevertheless, CT angiograms are still believed to be

superior to MRI angiograms for visualizing vascular

abnormalities.


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Some groups have also suggested MRI-guided cathe-

terization and real-time MRI-guided catheterization allow

the assessment of an impressive list of direct and calculated

parameters, namely the construction of RV pressure vol-

ume loops, RV afterload, myocardial contractility, pump

function, and RV–PA coupling, which are all useful to

assess PAH severity and response to therapy [46, 105].

Using both RHC and cardiac MRI, some have estimated

RV power. Indeed, RV mechanical efficiency is lower in

NYHA FC III than II, thus correlating with PAH severity.

Decreasing mechanical efficiency of the RV is a charac-

teristic of deterioration of RV functions in iPAH. This team

has also assessed glucose uptake by PET scan. However,

the reduction of RV mechanical efficiency is not due to a

metabolic shift to glucose oxidation, as assessed by PET-

scan [106].

Unfortunately, the relationship between MRI measures

and pulmonary hemodynamics is less straightforward.

Previous studies have suggested that PAP correlates with

RV mass [52], Fulton ratio [52], RV wall thickness, PAAT

[52], pulmonary blood flow transit time [107–109], and

septal curvature (r = 0.77). If leftward ventricular septal

bowing is observed, systolic PAP may be expected to be

[67 mmHg [46]. Among different MRI-derived parame-

ters, namely PA areas, PA stain, average velocity, peak

velocity, acceleration time, and ejection time, it has been

determined that average velocity best correlates with mean

PAP reference. This may imply that RHC might lose its

prominence in the future [46]. However, significant RV and

septal thickening (as seen in long-standing PAH), low left

ventricular systolic pressure may also influence the

importance of the septal curvature [52]. Similar correla-

tions have been suggested for pulmonary vascular

resistance [110]. However, these conclusions should be

considered with caution as these correlations were not

observed by other teams [111].

Followup values

The accuracy and reproducibility of cardiac MRI in

assessing cardiac functions and morphology makes it a

powerful tool for treatment follow-up [90]. Indeed, changes

in MRI-derived measurements are relevant in PAH. For

instance, a progressive dilation of RV, and a decrease in

LV systolic volume and RV stroke volume as detected at a

1-year follow-up by MRI have been correlated with a

worse prognosis in PAH [52, 96]. Changes in RV stroke

volume overtime also correlated with changes in exercise

capacity [112]. Changes in myocardial perfusion, RV mass,

and interventricular septal shift were also observed with

therapy [8, 46, 113, 114].

Limitations

The main limitation of MRI is imposed by the core phys-

ical principle of this technique, i.e. its incompatibility with

metal compounds such as the delivery pump for continuous

parenteral therapies commonly used in PAH [8]. Never-

theless, some teams have extended patients’ tubulure,

allowing the pump to be left outside the MRI room [8].

Moreover, MRI remains expensive, not widely available,

and time-consuming compared to CT scan [90]. MRI is

also problematic for claustrophobic patients (i.e. 2.3% of

patients subjected to MRI), although it can easily be cir-

cumvented by light sedation and the development of a

short-bore machine reducing the noise and the close-space

feeling [115, 116]. As for other imaging technics, body

movements, as well as respiratory and cardiac motions may

result in artifacts. Fortunately, technical improvements in

acquisition speed have reduced breath-holding and scan

times [52]. Finally, end-stage renal disease has been

associated with cases of nephrogenic systemic fibrosis

when gadolinium-containing contrast agents are used [90].

Due to its availability and ease of use, echo will probably

remain the tool of choice for first-tier screening and

detection, whereas MRI will be used subsequently to assess

lung and heart functions [52].

Conclusion

Imaging techniques are improving very fast, and, alone or

in combination, allow the measuring of an increasing

number of clinically relevant parameters both at baseline

and during follow-up. Although RHC is still the gold

standard to establish a diagnosis of PAH, novel imaging

tools are coming progressively closer to the same diag-

nostic quality. While some standardization of procedures is

required to reduce inter-observer and inter-site variability,

imaging techniques are very useful to support a diagnosis

of PAH diagnosis, to exclude other causes of PH, and to

objectively assess disease severity.

Circulating biomarkers

In parallel with imaging techniques, circulating biomarkers

are intensively explored by several groups. Ideally, blood

and urine biomarkers that (1) become abnormally elevated/

decreased in early or severe/end-stage disease (depending

on the biomarker), (2) are independent of indirect conse-

quences of PAH (e.g., renal and left ventricle function); and

(3) parallel the progression of the disease or the favorable

response to therapeutic interventions. Unfortunately, no such


123

biomarker has yet been identified. Nonetheless, some of

those presented below show some interest and despite the

fact that no perfect biomarker is yet available, a combi-

nation of several biomarkers might be sufficiently accurate

for diagnostic or prognostic purposes.

Cardiac-derived biomarkers

Brain natriuretic peptides (BNP and NT-proBNP)

Plasma brain natriuretic peptide (BNP) and its biologically

inactive N-terminal fragment, NT-proBNP [117], are car-

diac hormones secreted by the cardiac myocytes and are

used as ventricular dysfunction biomarkers [118]. In PAH,

BNP and NT-proBNP, levels increase due to enhanced

synthesis by the RV and reflect the RV structure and

function [117, 119, 120]. Both forms appear to predict

survival in idiopathic PAH (iPAH) [117, 119, 121, 122] as

well as other types of PH [123, 124]. Furthermore, natri-

uretic peptide levels parallel changes in pulmonary

hemodynamics overtime [253]. However, natriuretic pep-

tides are not a specific PAH biomarker since they are

largely influenced by renal function, fluid retention and

diuretics, situations frequently encountered in PAH [125,

126].

Troponin T

Cardiac troponins are regulatory proteins that can be

detected by highly sensitive assays in peripheral blood after

their release, as a result of cardiac myocyte membrane

disruption. Torbicki et al. have evaluated the prognostic

value of that biochemical parameter in patients with severe

PH [127]. Detectable troponin T was associated with a

higher heart rate and NT-proBNP levels, a shorter 6-min

walk distance, and a poor prognosis. Indeed, 63% of

patients with detectable levels of cardiac troponin T died

during the 2-year follow-up. Although troponin T is a

marker of disease severity, and of prognostic value, it rises

only in end-stage disease [38, 127] and may be confounded

by left heart disease and renal impairment [128–130].

Osteopontin

Osteopontin (OPN), being a cytokine, is related to the

inflammatory process. Circulating OPN has been shown as

an independent predictor of survival in iPAH patients, and

correlates with severity [131]. Indeed, OPN is significantly

higher in iPAH patients compared to healthy subjects. In a

retrospective study, baseline level of OPN has been shown

to correlate with 6-min walk distance, mean PAP, and FC.

However, the origin of OPN production is still unclear. It is

hypothesized to be produced by the RV but this still has to

be confirmed [131]. Moreover, its biomarker value has to

be confirmed in larger studies.

Endothelium-derived biomarkers

cGMP

Cyclic guanosine 30,50-monophosphate (cGMP) is pro-

duced by the activation of guanylate cyclase and is

considered as an intracellular second messenger of natri-

uretic peptides, bradykinin and nitric oxide (NO) [132].

cGMP levels are significantly elevated in PAH patients’

urine compared to healthy controls, and inversely correlate

with the hemodynamic severity of PAH [133]. Wiedemann

et al. have measured cGMP levels in PAH and PH patients,

and, besides a marked increase in cGMP and ANP levels in

these patients, found that iloprost (a stable prostacyclin

analog) inhalation caused a decrease in cGMP in parallel

with pulmonary vasodilation and hemodynamic improve-

ment [134]. However, due to sizable interindividual

variations in cGMP levels, this biomarker has poor dis-

criminative properties [135].

Microparticles

Circulating microparticles (MP) are submicron membrane

fragments shed from activated or damaged vascular cells,

and released during apoptosis and/or activation of various

cell types [136]. Circulating endothelial MP have been

described as markers of endothelial injury as well as sys-

temic vascular remodeling [137]. Amabile et al. [138] have

shown that levels of circulating endothelial platelet endo-

thelial cell adhesion molecule (PECAM?), VE-cadherin?,

E-selectin?, and leukocyte-derived MP levels were

increased in PH patients compared to healthy individuals.

More specifically, levels of PECAM? and vascular endo-

thelium cadherin in PAH were significantly correlated with

the hemodynamic severity of PAH, suggesting that endo-

thelial MP might be reliable predictors of disease severity

in PH. Corroborating these results, Bakouboula et al. have

shown that MP containing active platelet tissue factor

(thrombokinase) and CD105 (endoglin) were increased in

patients with PAH compared to the control group [139].

Pro-coagulant MP were also linked to PH severity. They

hypothesized that circulating MP in the pulmonary vascu-

lar bed might also contribute to lung injury via diverse

pathways: impaired perfusion, vascular remodeling, leu-

kocyte recruitment, and inflammatory response. MP

containing active platelet tissue factor and CD105 may

possibly be considered as PAH early biomarkers in the near

future provided that other studies confirm the above find-

ings and further characterize these MP and their

involvement in PAH.


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Given these encouraging results, research is still ongo-

ing on MP in animal models to better assess their role and

their potential as biomarkers. In order to characterize cir-

culating MP during hypoxic PAH and to study their effects

on endothelial function, the study of Tual-Chalot et al. has

provided evidence that hypoxic circulating MP induce

endothelial dysfunction in rat aorta and PA by decreasing

NO production [140]. Furthermore, they showed that MP

display tissue specificity.

Platelet-derived MP may influence vascular function via

their involvement in leukocyte adhesion, thrombus for-

mation and interaction with endothelium. The role of

platelet MP in thromboxane A2 (TXA2) production was

examined in rabbit PA [141]. TXA2 is well known to be

raised in atherosclerosis, which is a chronic vasculature

disease in which oxidative stress plays a key role [142].

That study provided evidence that platelet MP act as a

cellular source of TXA2 in rabbit aorta and PA. Yet, this

experimental approach demonstrated the ability of platelet

MP and endothelial cells to participate in the transcellular

conversion of arachidonic acid to the important vasocon-

strictor TXA2. Because TXA2 is an interesting mediator in

hypertension known to be deregulated in human PAH

[143], that work has provided an insight into the role of

platelet MP in vascular tone control.

Plasma von Willebrand factor

Plasma von Willebrand factor (vWF) is a large glycopro-

tein synthesized mainly in endothelial cells. The presence

of dysfunctional endothelial cells in PAH has been sug-

gested to be due to increased proteolysis and vWF release

[144]. Veyradier et al. demonstrated that baseline vWF

levels and vWF proteolysis were increased in ten patients

with severe PAH [144]. Moreover, these alterations were

reversible upon initiation of continuous epoprostenol

infusion, and paralleled the improvement of hemodynamic

measurements. More recently, Kawut and coworkers

showed that increased vWF levels at baseline and follow-

up were associated with worse survival in a cohort of 66

PAH patients [145].

D-dimer

D-dimer is a specific marker of cross-linked fibrin and is

associated with microvascular thrombosis, an important

component of PAH pathophysiology. D-dimer, as mea-

sured with an ELISA method, is increased in PAH patients

and correlates with patients’ functional capacity and

hemodynamic severity in iPAH [146, 147]. On the other

hand, such correlation was not found in PAH associated

with systemic sclerosis [148]. These data may suggest that

microvascular thrombosis might not play an important role

in the pathogenesis of PAH in patients with systemic

sclerosis, or that microvascular thrombosis occurs in the

systemic circulation independently of the pulmonary vas-

cular remodeling in these patients. Moreover, D-dimer

elevation occurs in diverse clinical situations, limiting its

accuracy in PAH.

Cancer-shared PAH biomarkers

Inflammation state

GDF-15 Growth differentiation factor (GDF)-15, which

belongs to the transforming growth factor (TGF)-super-

family of cytokines, was identified as an independent

predictor of long-term mortality in iPAH [149]. That bio-

marker displays a tight correlation with echo-measured RV

systolic pressure NT-proBNP plasma levels in systemic

sclerosis-associated PAH. These results suggest that GDF-

15 may have value as a biomarker, but larger studies on

PAH patients are obviously required at this stage [150,

151].

Endothelin-1/endothelin-3 Endothelin-1 (ET-1) is a pro-

liferative cytokine and a potent endogenous vasoconstrictor

with remodeling properties. Plasma ET-1 levels have been

shown to be increased in PH patients [152–155], and ET-1

protein as well as mRNA expression are enhanced in

endothelial cells of affected vessels [156]. More impor-

tantly, synthetic ET receptor antagonists (e.g., bosentan)

were shown to have beneficial effects in patients with

various forms of PAH [157–159].

Rubens et al. [153] have found that active ET-1 and its

precursor, big ET-1, correlate with disease severity and are

good prognostic markers for PAH patients. However, ET-1

measurement is not easily achieved and encounters tech-

nical difficulties such as the extremely short half-life of the

peptides (a few minutes) and their sensitivity to physio-

logical and pathological factors [160]. Endothelin-3 (ET-3)

is produced in many organs by various cells, including

endothelial cells. Montani et al. [161] have demonstrated

that the ET-1/ET-3 ratio is increased, whereas ET-3 plasma

concentrations are decreased in PAH. ET-1 and ET-3 levels

were correlated with hemodynamic and clinical markers of

disease severity, suggesting that the ET-1/ET-3 ratio might

be a novel prognostic factor in PAH.

LIGHT Otterdal et al. [162] investigated thrombus for-

mation and inflammation in PAH pathogenesis. They

demonstrated that lymphotoxin-like inducible protein that

competes with glycoprotein D for herpes virus entry

mediator on T lymphocytes (LIGHT) serum levels, a

platelet-derived ligand of the tumor necrosis factor super-

family, were increased in PAH compared to control

subjects. Moreover, LIGHT levels are significantly related


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to mortality in PAH patients. These findings suggest that,

since the prothrombotic effects of LIGHT in PAH involve

endothelium-related mechanisms, these effects might be part

of a common terminal pathway involved in PAH pathogene-

sis. However, further evidence is needed to confirm these

hypotheses, and larger trials must be carried out before

LIGHT can be considered as a reliable biomarker [162].

C-reactive protein C-reactive protein (CRP), a marker of

inflammation and tissue damage, has been demonstrated to

be an efficient outcome predictor in PAH. Indeed, its level

is significantly higher in PAH patients compared to control,

independently correlating with severity and predicting

mortality and clinical worsening. Moreover, normalization

of CRP levels with therapy was associated with improve-

ment in functional capacity, cardiac index and long-term

survival [163, 164].

Oncogenes/tumor suppressors

A growing number of cancer hallmarks turn out to be

present in PAH. Our recent review [165] reports the ther-

apeutic value of proto-oncogenes/tumor suppressor

proteins. These therapeutic targets might also have bio-

marker potential. Examples of the activation of oncogenic

pathways in PAH include the increase in cytokines and

growth factors such as epidermal growth factor (EGF),

PDGF, cytokines (e.g., IL-6) [166], or peptide agonists

such as ET-1 [167–170]. These factors can activate bio-

logical pathways such as proliferation, migration, survival

and metabolism switches. Some cytokines are already

considered as cancer biomarkers [171–174]. One might

surmise that a cytokine such as IL-6 may also have value as

a PAH biomarker. The main issue to be addressed to assess

that hypothesis is the lack of specificity of many of these

factors. However, cancer and PAH are easy to differentiate,

given their different symptoms, which would bring more

value in using proto-oncogenes as PAH biomarkers.

Pim1 proto-oncogene A preclinical trial is currently

ongoing to assess the potential of the Pim1 protoconcogene

as a biomarker in PAH. As explained earlier, our current

understanding of PAH physiopathology relies on NFAT

and STAT3 activation. Thus, our group has shown that

STAT3 is responsible for enhancing not only NFAT but

also Pim1 expression [20]. In fact, the latter oncogene is

required for NFAT activation in PAH. This means that, in

PAH, NFAT cannot be activated in the absence of Pim1

and cannot deregulate Ca2? homeostasis and other mech-

anisms responsible for PASMC proliferation and resistance

to apoptosis found in distal PA.

Interestingly, Pim1 is not normally expressed in human

tissues. Although Pim1 is also found in diverse types of

cancer, Paulin et al. showed that Pim1 exhibits high specificity

for the pulmonary vascular remodeling that characterize PAH

[20]. In PAH, Pim1 is markedly overexpressed in the

pulmonary vascular bed. Moreover, the activation of these

pathways is also observed in circulating immune cells,

possibly due to the inflammatory state of the lung. Indeed,

our group has shown that it is possible to measure Pim1

expression in the buffy coat of PAH patients. More impor-

tantly, preliminary data suggest Pim1 blood levels are

elevated in early disease and strongly correlate with the

severity of the disease. Finally, the simplicity of the method-

ology used to measure Pim1 (from blood sample and urine)

supports Pim1 as a promising biomarker in PAH [20].

Other oncogenes/tumor supressors The p27 tumor sup-

pressor is often downregulated in cancers harboring p53

mutations. Interestingly, overexpression of p27 decreases

PASMC proliferation and prevents hypoxia-induced PH in vivo

[175, 176]. Conversely, p53 deficiency in a hypoxia mouse

model is associated with a more severe PAH, an increase in

HIF-1a, and a loss of p21 expression. Furthermore, that tumor

suppressor has been shown to have a protective role in a left-to-

right shunt model, which strengthens its protective effect [165].

Yang et al. aimed to investigate the role of apoptosis in the

PA remodeling of PH secondary to hypoxia and to determine

the relative expression of selected genes [177]. Compared to a

control group, the apoptotic index of the hypoxic group

decreased significantly. Through the methods of in situ

hybridization and RT-PCR, this group found that Bcl-2

expression was increased, whereas Bax was decreased

significantly in the hypoxic group. The alteration in Bcl-2

and Bax expression induced by hypoxia plays an important

role in PA remodeling, which is the main pathological change

found in PH secondary to hypoxia [177].

Current understanding of cancer pathophysiology in

apoptosis resistance is fundamental to develop efficient

treatments, which might also be of help in PAH treatment.

However, a number of studies are required to evaluate

whether these oncogenes/tumor suppressors are quantifi-

able and have biomarker values. Indeed, a compounding

factor is that these proteins are not secreted. Actually,

unlike with cancer where the tumor is usually removed and

in situ hybridization may be performed, there is no direct

way to extract these polypeptides with PAH. Nonetheless,

it may be thought that, as for Pim1, some of these factors

might be found in circulating immune cells and, thus, in

patient’s buffy coat. Moreover, proteomic technology and

future improvements in microsurgery might facilitate the

investigation of oncogenes/tumor suppressors in PAH.

miRNA

Small noncoding miRNA (21–23 nt) are now recognized as

important regulators of gene expression and are involved in


123

most physiological and pathological processes. Several

studies are currently assessing whether miRNA expression

in blood samples might be used to identify PAH progres-

sion and to help in its diagnosis. Indeed, miRNA can be

secreted and may thus be found in fluid samples. Further-

more, as already discussed, inflammatory mediators are

important actors in PAH. Our laboratory has demonstrated

that the pathways involving NFAT, STAT3 and miR-204

described in PASMC (see below) may also be found in

inflammatory cells, since altered levels of mRNA and

oncogenes have been demonstrated in PH patients’ buffy

coats [14, 20].

Tumor suppressor miR-204 Our group has characterized

the crucial implication of miR-204 in PAH pathogenesis. In

fact, STAT3 is responsible for the decreased expression of

miR-204, which is itself a STAT3 inhibitor. In PAH, the

increase in circulating factors activates STAT3, and,

therefore, downregulates miR-204. Furthermore, as said

earlier, STAT3 enhances NFAT expression. Thus, via

many pathways previously described [15, 178], NFAT

leads to the proliferative and anti-apoptotic phenotype that

is responsible for the vascular remodeling caused by PAH.

This feedback loop between STAT3 and miR-204 might

explain the resistance to inhibitors of circulating factor

receptors such as imatinib (a tyrosine kinase inhibitor) and

bosentan (an endothelin receptor antagonist). We have

described the therapeutic value of miR-204 restoration,

since nebulization with a miR-204 mimic reverses the

pathology in the monocrotaline-PAH rat model [19]. The

latter study also highlights the biomarker potential of miR-

204. Indeed, the decrease in miR-204 expression is found

specifically in the lung and in PAH models, and correlates

with PAH severity in human buffy coat. Thus, although

these results must be replicated in larger patient cohorts,

miR-204 represents a promising biomarker that also cor-

relates with disease severity.

miR-21 oncogene As previously mentioned, PAH is

characterized by an increase in circulating blood factors. A

noteworthy observation is that TGF and bone morphoge-

netic protein (BMP) stimulation have been shown to

rapidly induce miRNA-21 (miR-21) [179, 180]. The latter

miRNA is significantly increased and correlates with poor

prognosis in lung cancer [181].

Wang et al. [182] described the profile of miRNA

expression in human arteries with arteriosclerosis obliter-

ans. Among them, miR-21 was mainly found in arterial

smooth muscle cells and was increased by [sevenfold in

arteriosclerosis obliterans, which was related to HIF-1aexpression. In cultured human arterial smooth muscle cells,

cell proliferation and migration were significantly

decreased by the inhibition of miR-21. These authors were

also able to confirm that tropomyosin-1 is a target of miR-

21 that is involved in the intracellular effects of miR-21.

The study of Sarkar et al. [183] has investigated the role of

miR-21 in hypoxia-induced PASMC proliferation and

migration. The latter group showed that miR-21 expression

increased by threefold in human PASMC after a 6-h

hypoxia (3% O2) and remained high (twofold) even after a

24-h hypoxia. They further showed that miR-21 is essential

for hypoxia-induced cell migration. Protein expression of

miR-21 target genes, i.e. programmed cell death protein-4,

Sprouty 2, and PPARa, was decreased by hypoxia and in

PASMC overexpressing miR-21 under normoxia, and was

increased in hypoxic cells in which miR-21 had been

knocked down. Their findings suggest that miR-21 plays a

significant role in hypoxia-induced PAMSC proliferation

and migration through the regulation of multiple gene

targets.

Using PAH rat models (hypoxic and monocrotaline),

Caruso et al. demonstrated miR-22 and miR-30 were

downregulated, whereas miR-322 and miR-451 were signif-

icantly upregulated in both PAH models. Interestingly,

miR-21 was selectively and consistently downregulated after

monocrotaline injection, but not under hypoxia. They also

described the downregulation of miR-21 expression in iPAH

human samples [184]. These findings suggest that a

substantial loss of miR-21 is associated with vascular

remodeling in monocrotaline-exposed lungs. The authors

suggested that reduced BMP signaling is possibly related to

miR-21 downregulation in the monocrotaline model, which

might be involved in the alteration of smooth muscle cell

phenotype found in PAH.

Taken together, these studies highlight the importance

of ascertaining the role of miR-21 in the different PAH

models as well as defining the potential effect of miR-21

modulation in disease prevention. Undoubtedly, these data

emphasize the importance of investigating miR-21 in the

pathology of PAH.

miR-210 oncogene miR-210 is the main hypoxia-sensi-

tive miRNA involved in the regulation of the hypoxic

response in tumor cells as well as tumor growth [185]. Its

expression is induced by HIF-1a and is known to protect

cancer cells from hypoxia-induced apoptosis, and to par-

ticipate in mitochondrial impairment and cell cycle

regulation, all features that are also found in PAH [19, 184,

186–188]. Nonetheless, miR-210 expression has not been

found to be altered either in human or PAH animal models.

miR-145 tumor suppressor The biomarker potential of

miR-145 has already been assessed in cancer [189].

miR-145 is the most abundant miRNA in normal vascular

walls, and is significantly decreased in pathological con-

ditions of vascular walls with neointimal lesions and

dedifferentiated vascular smooth muscle cells. Cell dedif-

ferentiation is well known to play a crucial role in the


123

pathological vascular remodeling process that occurs in

several vascular pathologies, including PAH. Differentia-

tion can be affected by several stimuli, including growth

factors typically increased in PAH. Furthermore, Cheng

et al. [190] demonstrated that its restoration leads to the

loss of the pathological phenotype in an in vitro model of

vascular smooth muscle cells as well as in an in vivo model

of carotid angioplasty in rats. Hence, it might be very

interesting to assess the biomarker value of miR-145 in

PAH as well to determine whether it is also reduced in

PAH patients’ buffy coat, and whether it correlates with the

severity of the disease.

Conclusion Being already considered as biomarkers in

cancer, miRNA might be potential biomarkers in PAH in

the near future. Since miRNA are very short sequences that

are present at low concentrations in the blood, extraction

inaccuracies as well as the variance of replicate values will

constitute a serious methodological challenge [191]. Stan-

dardization of sample conservation, technical dosage and

extraction are of utmost importance before translating the

profiling of serum miRNA to clinical practice, lest such a

promising and powerful tool be misused.

Metabolic biomarkers

Serum lactate dehydrogenase (LDH) The Warburg effect

is increasingly correlated with aggressiveness and poor

prognosis in cancers [192]. Given that a similar metabolic

disorder is also found in PAH, serum LDH might be a

biomarker, although not specific, of the Warburg effect in

PAH.

Urine metabolomic derived biomarker A few teams are

currently working on the use of metabolomics-derived

biomarkers able to differentiate cancer from healthy

patients, and correlating with severity. These groups have

measured various metabolites in patients’ urine such as

quinolinate, 4-hydroxybenzoate, and gentisate [193]. It

might be of major interest to investigate whether specific

differences are observed in the urine of PAH patients.

Oxidative stress biomarkers and PAH

Reactive oxygen species (ROS) are predominantly impli-

cated in cell damage via the inhibition of protein, lipid and

DNA normal functions. These highly reactive species may

initiate several peroxidative reactions damaging the genetic

cell apparatus, as well as cell membranes, proteins and

lipids. ROS may be produced in the lung tissue in severe

PH patients as a result of tissue hypoxia [194], ischemia

[195] or via inflammatory cascade activation and increased

production of inflammatory cytokines [196, 197]. Oxida-

tive stress occurs when repeated external insults result in

excessive ROS formation, which overwhelms antioxidant

systems, thus creating an altered redox state favors oxi-

dation. Oxidative stress is also considered as a cancer

hallmark, but the present discussion will focus on the role

of ROS markers in PAH.

Increases in oxidative stress and in lipid peroxidation

products are associated with elevated PA pressure in dif-

ferent animal models of PH [198–200]. Altered ROS

generation is believed to play a crucial role in the vascular

responses observed under hypoxic conditions, such as

deregulation of pulmonary vasomotor tone [201–203].

Moreover, some animal models studies have shown that

ROS play a role in pathological RV remodeling. It is well

established that increased PAH causes pressure overload of

the RV, leading to RVH [204, 205]. Increased ROS gen-

eration and altered redox state are some of the critical

features of the transition from hypertrophy to heart failure.

Among various causes of hemodynamic and structural

abnormalities of the decompensating RV are neurohor-

monal signaling (angiotensin-II, ET-1, aldosterone),

natriuretic peptides, and inflammation, but also oxidative

stress (including ROS and reactive nitrogen species) [206].

Uric acid

Uric acid is an endogenous free radical scavenger pro-

tecting cells from ROS and reactive nitrogen species

damage. It has been demonstrated that uric acid levels are

increased in PAH [207]. Voelkel et al. hypothesized that

the site of uric acid production in PAH may be either the

ischemic lung tissue or the ischemic RV, or both [207].

Uric acid serum levels correlate with hemodynamic and

functional parameters [208, 209], and are related to sur-

vival in adult [33, 208] and pediatric [209] PAH. Uric acid

is thus a biological serum marker of disease severity in

PAH. However, uric acid serum levels are influenced by

allopurinol, diuretics and hypoxemia and display signifi-

cant interindividual variability, limiting its accuracy as a

biomarker in PAH.

Isoprostanes

Isoprostanes are products of the peroxidative attack of

membrane lipids and are present at nanomolar concentra-

tions in the blood of normal individuals [210, 211]. In

several disease states, and particularly pulmonary diseases

such as PAH, isoprostane levels are increased [199, 212].

Moreover, given their potent and multiple biological

activities, isoprostanes have been thought to account for

many of the pathophysiological processes found in PAH

(e.g., decrease in endothelial relaxing factors) [213]. A key

question about isoprostane analysis is which isoprostane

should be analyzed among the 64 distinct isoprostanes that


123

can be generated from arachidonic acid [214]. The 5- and

15-series F2-isoprostanes are produced in closely equal

amounts in vivo, whereas the 8- and 12-series F2-isopros-

tanes are produced in lower yields [215]. Considering their

relative stability, the availability of inexpensive assays for

quantification, the easy collection of blood and/or urine

samples, and the direct relationship between isoprostane

accumulation and oxidative stress intensity has made these

‘‘markers’’ of oxidative stress optimal biomarkers of lung

disease states [213]. Hence, isoprostanes might also be

potentially useful markers for PAH.

Serotonin (5-HT)

An elevation of circulating peripheral serotonin (5-HT) has

been shown to occur upon PAH development both experi-

mentally and clinically [216, 217]. This vasoactive amine may

also produce ROS [218–220] and can induce protein car-

bonylation in PASMC [221]. Moreover, 5-HT stimulates

PASMC proliferation, PA vasoconstriction and local micro-

thrombosis [222]. In addition, studies have demonstrated that

inhibition of 5-HT receptors and 5-HT transporters is able to

delay PAH development and extend rat survival [223, 224].

The therapeutic value of 5-HT also highlights its interest as a

biomarker. Thus, given the evidence that PAH patients have

increased plasma 5-HT levels, and that this hormone may be

considered as a pro-oxidant, it represents an attractive and

easily assayed biomarker for PAH diagnosis and follow-up.

Derived nitric oxide products

NO may act as a signaling molecule, a toxin, and a pro-oxidant

as well as a potential antioxidant [225]. It has been proposed to

act as a pro-oxidant when reacting with superoxide anion

(O2-), thereby generating peroxynitrite [226], or by itself at

high concentrations [227]. NO is a pulmonary vasodilator

produced and released by the endothelium. Among its main

functions are vascular tone regulation [160], platelet aggre-

gation and inhibition of vascular smooth muscle cells

proliferation. A reduction in available NO occurs in ROS-

overproducing cells. The alteration of eNOS expression has

also been associated with systemic and pulmonary hyperten-

sion [228–230]. In theory, low plasma NO might be a marker

of endothelial dysfunction and possibly PAH. However, NO is

too unstable to be measured in gaseous form in the blood

[160]. However, NO treatment has already been tested,

without much success, but NO-products and NO itself may

help PAH diagnostic and follow-up.

HIF-1

HIF-1, which contributes to oxidative stress, is involved in

the PAH pathogenesis, raising a genuine interest in this

protein. HIF-1 is a transcription factor responsible for

mediating physiological responses to hypoxia, and is found

in a variety of organisms. The HIF-1a subunit, also called

‘‘oxygen sensor’’, may be accumulated under normoxic

conditions as a result of various stimuli such as ROS,

growth factors, oncogenic activation and NO [231, 232].

The effect of NO on HIF-1a expression is reciprocal and

depends on the chemical structure and concentration of the

NO donor [233]. HIF-1 alteration may also indicate oxidant

damage [198], and, thus, both in vitro and in vivo studies

have demonstrated that HIF-1 protein levels are inversely

proportional to O2 levels in cell media [234, 235]. Fur-

thermore, HIF-1 has long been believed to increase ‘‘early-

response gene’’ transcription, which modulates vascular

remodeling in the lung as well as cardiovascular function

[236, 237]. Thus, HIF-1 is a promising candidate as a novel

biomarker and/or as a target for PAH treatment. Moreover,

its pathway actors should be carefully studied in order to

find their biomarker potential.

NADPH oxidase/superoxide dismutase (SOD)

Using monocrotaline PAH model, Redout et al. have

demonstrated that NADPH oxidase and mitochondria-

derived ROS production increase RV failure [204]. Their

findings indicate that mitochondrial SOD-1 and SOD-2

mRNA levels were decreased, and nitrotyrosine staining

confirmed the presence of oxidative stress. Importantly,

Archer et al. have identified mitochondrial SOD-2 meth-

ylation as a potential epigenetic mechanism for PAH in the

Fawn-Hooded rat PAH model [238]. It may be relevant to

investigate whether SOD-2 is also downregulated in human

PAH. Interestingly, SOD-2 epigenetic downregulation

impaired H2O2-mediated redox signaling, activating HIF-

1a and creating an apoptosis-resistant state. Importantly,

SOD-mimetic therapy reversed PAH in vivo, reinforcing

the involvement of HIF-1. The study by Archer et al. also

demonstrated that an increase in SOD-2 can restore mito-

chondrial function, inhibit proliferation and increase

PASMC apoptosis in vitro. This study was the first dem-

onstration of an epigenetic basis for PAH. These studies

demonstrate that SOD-2 might be a powerful tool for PAH

treatment and might have potential diagnostic value in

PAH in the future, directly or via proteins functionally

related to SOD-2.

Conclusion

There is an urgent need for alternative biomarkers that are

safer and more reliable than those that are currently

available. Because ROS may promote vasoconstriction,

vascular smooth muscle cells proliferation and vascular

remodeling in many PH forms, research should be pursued


123

towards a thorough understanding of its critical role. There

is mounting evidence of a cross-talk between PAH patho-

genesis and oxidative stress, and monitoring oxidative

stress signs and damage may help to improve PAH diag-

nostic and prognosis. Additionally, these data may provide

a rationale for developing therapeutic strategies in RV

failure due to PAH.

Other biomarkers

A renal-derived biomarker: creatinine

Renal dysfunction is well known to predict mortality in

many cardiovascular diseases. Not surprisingly, Shah et al.

demonstrated that higher serum creatinine levels and

decreased glomerular filtration were associated with

hemodynamic severity and worse survival in PAH, results

that were also reported by other groups [126, 164].

CNS-derived: sympathetic nerve activity

Velez-Roa et al. [239] showed that PAH patients had

increased muscle sympathetic nerve activity compared to

controls, especially in advanced PAH. Moreover, they

demonstrated that sympathetic hyperactivity was partially

chemoreflex-mediated. Similarly, McGowan et al. docu-

mented muscle sympathetic nerve activity burst frequency

was higher in PAH patients and was inversely correlated

to the low-frequency spectral component of heart rate

variability [240]. The authors suggested that reduced heart

rate variability and sympathetic nervous system excitation

in PAH patients might increase the likelihood of sudden

death or accelerate progression to RV failure. This

hypothesis was supported by recent experiments docu-

menting the relationship between muscle sympathetic

nerve activity and disease severity and prognosis [254].

This deserves prospective evaluation in larger cohorts in

order to consolidate the status of heart rate variabil-

ity spectral power and sympathetic hyperactivity as

useful noninvasive surrogate markers of disease severity

in PAH.

Proteomic-derived biomarkers

Improvement in proteomic tools has led to the assessment

of proteomic-derived PAH biomarkers. Indeed, Zhang

et al. have suggested that simultaneous analysis of the

expression of ten protein spots may distinguish patients

with PAH from normal controls [241]. Nine proteins and

their isoforms were significantly different in the serum of

PAH patients relative to controls, including leucine-rich a-

2-glycoprotein, haptoglobin precursor, albumin isoform-2,

transferrin variant, C3 complement, hydroxypyruvate

reductase isoform-1, RAF1, fibrinogen isoform c-A, and

fibrinogen isoform c-B. In addition, significant and

important associations between LRG levels and functional

class FC (r = 0.71, p \ 0.01) and cardiac output (r = -

0.65, p \ 0.01) were shown.

Geraci et al. have also hypothesized that the gene

expression pattern found in PAH patient lung tissue has a

characteristic profile compared to healthy lungs [242].

Using oligonucleotide microarray technology, they char-

acterized gene expression patterns in the lung tissue

obtained from six PAH patients—including two patients

with the familial PAH form—as compared to six matched

healthy patients. All PAH lung tissue samples had a

decreased expression of several genes encoding various

kinases and phosphatases, whereas the expression of sev-

eral oncogenes and genes encoding ion channel proteins

was increased. Alterations in the expression of TGF-breceptor III, BMP2, MAP kinase kinase-5, RACK-1, apo-

lipoprotein C-III, and laminin receptor-1 were found

exclusively in PAH patients. The microarray gene

expression technique is a very useful molecular tool pro-

viding novel pertinent data that may bring a better

understanding of the pathobiology of different PH clinical

phenotypes. Unfortunately, the methodology used in such

studies cannot yet be used in the clinical practice due to its

high cost. Nevertheless, it remains a promising new

approach that should be kept in mind with the incessant

technical improvements in proteomic analysis, and

might also lead to the discovery of more easily assessable

serum or urine biomarkers. Taken together, these results

indicate that proteomic analysis might be helpful in PAH

diagnosis.

Red blood cells distribution width

Red blood cell distribution width (RDW) reflects red blood

cell size variability and is commonly reported in automated

complete blood counts. The relationship between RDW

and PAH was investigated in a prospective study of 162

patients [243]. The authors found RDW to be related to

disease severity and demonstrated that it is independently

associated with mortality in PAH. Moreover, RDW per-

formed better as a prognostic indicator than NT-proBNP

and added important prognostic value over NT-proBNP

measurement and exercise capacity. The authors concluded

that RDW and NT-proBNP might be used in combination

to improve PAH prognosis.

Immune system-derived biomarkers: antibodies

Specific antibodies such as anti-Scl-70 may also be used as

biomarkers to assess the implications of other pathologies

[45]. Around 40% of PAH patients have positive but low


123

antinuclear antibody titers [244]. Other antibodies seem to

be promising, such as anti-endothelial cell antibodies [245]

or even antifibroblast antibodies [246]. Indeed, considering

the ease with which antibodies can be tested, it might be of

great interest to assess the presence and level of such

antibodies in the serum of PAH patients. This might rep-

resent easily measurable clinical parameters of potential

usefulness for future diagnosis and prognosis.

Hyponatremia

Growing evidences demonstrate that hyponatremia is

strongly associated with advanced RV dysfunction, poor

survival and WHO FC in PAH [247]. Since it is already

used and it is easy to assess, hyponatremia could represent

a powerful and independent biomarker. However, the

mechanism of hyponatremia remains elusive. Thus, even

though renal dysfunction is frequently seen in PAH patients

often treated with diuretics, Forfia et al. hypothesized that

hyponatremia may be a result of neurohormonal activation

(increased in PAH) possibly caused by more advanced RV

dysfunction [247].

High-density lipoprotein cholesterol (HDL-C)

It is well known that HDL-C is associated with poor

prognostic in cardiovascular diseases, but, interestingly,

decrease in this factor is also associated with higher mor-

tality and clinical worsening and outcome in PAH.

Furthermore, Heresi et al., have demonstrated that a

decrease in HDL-C does not appear to be caused by

underlying cardiovascular risk such as diabete mellitus,

BMI or other cardiovascular risk factors. Moreover, HDL-

C level is even lower in PAH patients than in a ‘‘high-risk

population’’ (patients with coronary disease, systemic

hypertension and diabetes, conditions associated with

lower HDL-C) [248].

Conclusion

All the biomarkers discussed in this part have been sum-

marized in Fig. 2. As shown above, current circulating

biomarkers may be helpful in the diagnosis of PAH, but

most of them lack specificity, and, unfortunately, many

appear only at severe stages of the disease. For these rea-

sons, PAH experts now consider the identification and

validation of a clinically relevant biomarker that is repre-

sentative of PAH physiopathology as a high priority

objective [89, 233, 249–252]. Cancer-related biomarkers

are promising tools but may lead to misdiagnosis. How-

ever, it should be kept in mind that, since symptoms of

cancer and PAH are very different, the use of cancer

biomarkers as PAH biomarkers might well be feasible in

practice. Moreover, the effectiveness of cancer diagnosis is

constantly improving. Likewise, oxidative stress-derived

biomarkers might be of some value for PAH diagnosis and

prognosis purposes. Finally, technological improvements

of proteomic tools may highlight the implication of new

actors in PAH physiopathology and lead to the discovery of

novel biomarkers and therapeutical targets. However,

many of the promising biomarkers already described must

still be assessed in larger patient cohorts to be validated. A

coordination between large PAH treatment centers should

be expected to include the standardization of study proto-

cols and tools in order to increase the number of patient in

such studies, and might significantly contribute to accel-

erate the validation of these biomarkers. In addition, the

combination of different biomarkers might be highly

relevant.

General conclusion

As of today, PAH diagnosis is still an exclusion diagnosis

involving a long period between the discovery of symp-

toms and diagnosis, resulting in a long delay before

initiating any treatment. Unfortunately, current imaging

and circulating biomarkers are only reliable in late stage

disease, limiting their utility for screening at-risk popula-

tions. Hence, improvements are much needed to improve

diagnostic strategies. Nevertheless, imaging biomarkers

represent crucial tools in determining the etiology of PAH,

excluding other types of PH and providing information

regarding RV function, disease severity and response to

therapy. The very rapid improvement in imaging tech-

niques seen in the previous decades strongly suggest that

imaging by CT, MRI and even echo will be further

improved, thereby decreasing the requirement for invasive

techniques such as RHC and allowing an earlier and more

accurate diagnosis. In addition, circulating biomarkers

show promises in PAH. Technical improvements are also

implemented in biochemical engineering, and the detection

of very small amounts of protein is now possible, thus

opening new avenues for circulating biomarkers. More

importantly, studies on biomarkers including oncogenes

and miRNA as well as oxidative stress-related proteins

may increase the understanding of PAH pathophysiology

and identify novel therapeutic targets.

Despite the technical improvements, the prospective

validation of new imaging and circulating biomarkers

relies on multicenter large-scale trials and standardization

of the techniques. While no ‘‘perfect biomarker’’ is likely

to be discovered any time soon, one might surmise that a

combination of biomarkers will together allow earlier


123

diagnosis, more accurate prognostication and improved

long-term outcomes in PAH.

Acknowledgments M.H.J. is supported by CAPES (Brazilian

Research Agency) and by Laval University. J.M. is a recipient of a

Graduate Scholarship from the Canadian Institutes of Health Research

CIHR) and A.C. received a graduate scholarship from La Societe

Quebecoise d’Hypertension Arterielle (SQHA). This work was sup-

port by Canada Research Chairs (CRC) and by Canadian Institutes of

Health Research (CIHR) to S.B. We would like to thank Dr. Richard

Poulin for editorial help in processing this manuscript.

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EDITORIAL

Clin. Invest. (2011) 1(3), 363–366New therapeutics for pulmonary arterial hypertension: do gene therapies have translational values?Roxane Paulin1*, Marjorie Barrier1* & Sebastien Bonnet†1,2

Keywords: gene therapy • pulmonary hypertension

Pulmonary arterial hypertension (PAH) is a devastating disease characterized by a marked and sustained elevation of pulmonary vascular resistance (PVR) increasing pulmonary arterial pressure. Progressively, patients will develop right ventricular hypertrophy (RVH) and then right ventricular failure. The pathogenesis of PAH is multifactorial. Endothelial cell (EC) dysfunction is well recognized as an early feature of the disease, leading to an imbalance in production of vasodilatator and vasoconstrictor factors (a decrement in vasodilatators such as prostacyclin and nitric oxide [NO], and a concomitant increase in vasoconstrictors such as thromboxane and endothelin-1) [1]. Over the past several years, therapy has focused on re-establishing this imbalance by, for example, exogenous delivery of vasodilatating prostaglandins and inhaled NO, or by blocking the endothelin axis, which leads to vasodilatation and improves the pulmonary circulation. Unfortunately, some of these treatments have undesired side effects, principally systemic hypotension (epoprostenol) and liver toxicity (sitaxentan, withdrawn from the market in 2010). Moreover, some patients are resistant to these therapies, and even for those who show responses, they failed to totally reverse PAH, unless the patients also received a lung/heart transplant. In fact, the major cause of the elevated PVR is the obstructive vascular remodeling due to an imbalance in the proliferation and apoptosis rates of the pulmonary artery smooth muscle cells (PASMCs) [1], which show a cancer-like behavior. Thus, the sci-entific community has started to focus more and more on the cellular and molecular mechanisms implicated in pulmonary artery remodeling and have started to develop therapies aimed at reversing the proliferative phenotype of PASMCs in the vascular wall [2]. Genomic approaches such as gene expression profiling and sequencing have demonstrated that many genes are aberrantly expressed in PAH [3] and are often involved in the activation of pathways responsible for the pro-proliferative and anti-apoptotic phenotype of PASMCs. Moreover, advances in gene-transfer technologies made the development of ‘gene therapy’ the modification of choice for therapeutic proposes. First designed to restore a genetic disorder or a mutation by transferring a normal copy of the gene, it soon became apparent that the range of targeted diseases could be extended to those showing aberrant gene expression. Therefore, in the last decade, several researches explored gene therapy for PAH.

Major recent advancesCompared with other thoracic malignancies, such as lung cancer, asthma, emphy-sema and cystic fibrosis, PAH appears to be late in gene therapy applications. In fact, the first clinical trial implicating gene transfer in PAH only began in 2010 [4]. Autologous endothelial progenitor cells (EPCs) programmed to overexpress the endothelial NO synthase (eNOS) were administered to patients with iPAH and

1Department of Medicine, Laval University, Quebec City, QC, Canada 2Centre de recherche de L’Hôtel-Dieu de Quebec, 10 rue McMahon, Quebec, Qc, G1R 2J6, Canada †Author for correspondence: Tel.: +1 418 525 4444 Ext. 16350 Fax: +1 418 691 5562 E-mail: [email protected] *Authors contributed equally.

363ISSN 2041-679210.4155/CLI.11.2 © 2011 Future Science Ltd

For reprint orders, please contact [email protected]

PAH associated with systemic sclerosis. Preliminary data showed modest but significant improvements in 6-min walk test and mean pulmonary arterial pressure. Given the fact that EPC therapy only (nonassociated with gene transfer) in two small-randomized trials in humans showed improvement in 6-min walk test and PVR [5], it is difficult to evaluate the real benefit of this additional gene therapy.

Antiproliferative candidates for gene therapy in PAHGene therapy targeting endothelial dysfunction is cur-rently being studied, but targeting the pro-proliferative phenotype of PASMCs is still to be achieved. Several experiments restoring mRNA levels of genes implicated in the aberrant activation of proliferative pathways in PAH have been performed in the last 5 years in animal models of PAH, and results are really promising.

■ BMPRII gene therapyHeterozygous mutations in BMPRII, a member of the TGF family of receptors, have been identified in many cases of familial and sporadic PAH [6]. In the case where BMPRII is not mutated, its downregulation is often observed in the PAH patients. Adenoviral deliv-ery of vector containing BMPRII gene in pulmonary vascular endothelium of chronic hypoxia-induced PAH (CH-PAH) rats reduced the pulmonary hypertensive responses [7]; whereas BMPRII intratracheal nebuliza-tion of adenoviral gene therapy in the monocrotaline rat model of PAH did not improve pulmonary hypertension despite a good distribution of the gene in the arteriolar network [8]. The heterogeneity of these results suggests that BMPRII may not be the best candidate for gene therapy. Nevertheless, even if BMPRII gene therapy could not be universally applied for PAH patients, it may be beneficial in some cases, or could be effectively coupled with other genes therapies.

■ Kv gene therapyContractility and proliferation of PASMCs is controlled by cytosolic Ca2+ levels, which are largely determined by membrane potential (Em). In fact, Em is depolarized in human and experimental PAH cells due to, at least in part, the decreased expression and function of voltage-gated K+ channels (Kv1.5 and Kv2.1). This ‘K+-channelopathy’ leads to PASMCs depolarization and Ca2+ overload, thus promoting vasoconstriction and PASMCs proliferation. Therefore, targeting and improving expression and function of these channels is thought to be promising. Restoration of Kv channel expression in PAH by aerosol gene therapy using an adenovirus expressing Kv2.1 might actually be beneficial [9]. Furthermore, Kv1.5 expression in established CH-PAH in rats reduces PVR and restores

RVH [10], and KCNA5 gene transfer in human PASMCs increases K+ currents and enhances apoptosis [11], demon-strating that Kv1.5 may serve as an important strategy for preventing the progression of PAH.

■ Survivin gene therapyPulmonary arterial remodeling in PAH might be explained by a pro-proliferative and antiapoptotic phenotype of the PASMCs. Survivin, a member of the inhibitor of apoptosis protein family, was first discov-ered in cancer. It is normally undetectable in healthy adult differentiated tissues, but it is expressed in remod-eled PAs from patients and rats with PAH, highlighting once again this proliferative phenotype. Adenovirus-mediated survivin overexpression induces PAH in rats, underlying an implication of this oncoprotein in PAH, whereas inhalation of a survivin-dominant negative adeno virus reverses established monocrotaline-induced PAH (MCT-PAH) [12] by avoiding PASMC proliferation and, thus, the subsequent PAH.

■ VIP gene therapyAmong its actions, vasoactive intestinal peptide inhibits proliferation of vascular smooth muscle cells. PASMCs treated with adenovirus expressing the VIP gene are less proliferative [13]. It could be interesting to see VIP-based gene therapy trials on animal models.

■ CGRP gene therapyCalcitonin gene-related peptide has also been described to have an antiproliferative effect. Intratracheal injec-tion of adenovirus carrying the pre-proCGRP gene into the lung of hypoxia-induced PAH mice attenuates the increase in PVR, RVH, remodeling and pulmonary pressure [14].

■ Growth factor gene therapyVEGF-A overexpression in MCT rats using cell-based gene transfer prevents PAH [15]. In the same way, HGF gene transfection in MCT rats prevents media wall thickening [16]. These findings imply that VEFA-A and HGF have protective effects against remodeling and could be good candidates for gene therapy.

Limitations of gene therapyGene therapies are hopeful treatments that might in the future help PAH patients. The aim of PAH treat-ment is to reduce pulmonary hypertension without affecting systemic circulation. Gene delivery into the lung via aerosol is one of these strategies. This could avoid many problems associated with systemic delivery by intravenous injection, such as immediate nuclease degradation and difficulty penetrating the endothelial barrier. Successful gene delivery via inhalation strongly

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depends on the development of advanced gene vectors. They must be able to protect the plasmid or sequence, provide a specific targeting site and effectively release these plasmids for the desired pharmacological effect. In the past two decades, numerous preclinical and clinical trials have been performed for thoracic malignancies, and lessons must be learned from these experiments.

First trials with adenovirus and adeno-associated viral vectors appeared to be ineffective and associated with detrimental immunologic responses and toxicity [17]. Recent work show some ingenious constructions, such as antibiotic coupling adenovirus or ‘helper-depen-dent adenovirus’ in which the proinflammatory virus sequences are missing. This permits a decreased inflam-mation associated with gene therapy. Lentiviruses are more efficient than other viral vectors but they need to be frequently readministered and lead to the same inflammatory problems.

Nonviral gene transfers are generally less efficient. In fact, plasmids as well as vectors used, require improve-ments. Some nucleotide sequences such as CpG motifs have been demonstrated to enhance proinflammatory responses [18] and have to be avoided in plasmid con-struct. Promoters, which regulate, in part, duration and tissue-specific localization of gene expression, have to be carefully chosen, particularly in the case of a desired restricted area of gene delivery (e.g., the vascular bed).

With the development of nanotechnologies, the emergence of new kinds of construction can be hoped for. Some nonviral vectors, for example liposomal ones, seem to cause less immunologic lung responses [19], which could worsen PAH. This is also true with nanomicelles. Formulations such as polyethylenimine (25 kDa, Sigma), cationic lipid 67 (GL67A, Genzyme corporation) or DNA nanoparticles (Copernicus), were shown to increase efficiency and duration of transgene expression.

Physical methods have also opened a new window in lung gene transfer improvement. Magnetic particles linked to DNA enhance a DNA response to magnetic fields (magnetofaction) and increase transfection rates in vitro. Unfortunately, application of this method in vivo is unsuccessful. Ultrasound (sonoporation) and electroporation have been shown to improve gene transfer in various tissues, but are not applicable in vivo because they are associated with lung damage such as hemorrhage.

Future perspectiveThe increased knowledge and understanding of stem cells has provided the scientific community hope to find cell therapies for PAH. Some are already used for genetic pathologies, mainly embryonic stem cells. The concept of using the patients’ own stem cells is strongly

emerging, avoiding ethical and immune issues. EPCs appear to be promising candidates, as described previ-ously. Mesenchymal stem cells are also very promis-ing [20]. The understanding of smooth muscle cell differ-entiation mechanisms are increasing and could be very useful for all vascular diseases, including atherosclerosis, heart failure and PAH.

The principle of coupling gene and cell therapies is also coming to the fore: isolate stem cells from a patient, make them express a gene of interest and then re-inject them into the patient. Coupling gene and cell therapy is already in trials using eNOS-transformed EPCs to treat PAH [4], and it could be speculated that targeting genes involved in proliferation and antiapoptotic mechanisms would be even more efficient. Furthermore, using mesen chymal stem cell-coupled gene therapy could be very promising considering their multipotency and their ability to accumulate at the site of tissue/organ damage and inflammation in vivo [21].

Targeting genes involved in the pro-proliferative and antiapoptotic phenotype by RNAi or siRNA could be one other possibility. As RNAi is increasly studied and better understood its use as a therapeutic is slowly emerg-ing. Aerosol coupling siRNA and transfecting agent would theoretically be able to silence the pathologically overexpressed gene(s) observed in PAH PASMCs. This is undoubtedly a promising concept to work on, even though technical issues remain to be resolved. siRNA lifespan, application modes and frequencies have to be characterized. The specificity, structural role and off-target effects also remain to be determined. Regarding the emerging concept of microRNA, which have a natu-ral RNAi action on genes and whose aberrant expression is implicated in human diseases and in PAH, we can also speculate that progress will continue in the next years. Administered mimics or antagomir in order to restore aberrant microRNA expressions could be another therapeutic intervention, but one that also requires improvements in transfectant agents.

ConclusionTreating PAH by gene therapy is very promising and targets and methods are being extensively studied. Treatments based on inhaled siRNA targeting different genes once a week or durable gene/cell therapies restor-ing one or few gene expression are achievable. Some technical issues remain to be solved and time is needed to realize the trials. Nevertheless, these therapeutics are associated with high technical costs. In this way, it will be difficult for these new treatments to be competitive against cheaper agents, such as dehydroepiandrosterone, dichloro acetic acid and trimetazidine [22]. These agents, already in trials, need much less expensive manufacturing costs for the relative same efficiency.

New therapeutics for pulmonary arterial hypertension: do gene therapies have translational values? EDITORIAL

future science group Clin. Invest. (2011) 1(3) 365

Financial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manu-script. This includes employment, consultancies, honoraria, stock ownership or options, expert t estimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

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