Natural History Study of Biomarkers in Pulmonary Arterial Hypertension

Introduction:

Pulmonary arterial hypertension (PAH) is a rare disorder associated with poor survival.
Endothelial dysfunction resulting from 1) genetic susceptibility, and 2) a triggering
stimulus that initiates pulmonary vascular injury, the two-hit hypothesis, appears to play a
central role both in the pathogenesis and progression of PAH. Inflammation appears to drive
this dysfunctional endothelial phenotype, propagating cycles of injury and repair in
genetically susceptible patients with idiopathic PAH (IPAH) and patients with
disease-associated PAH. However, despite mounting evidence of vascular inflammation in
patients with PAH, detailed phenotypic studies are lacking on the temporal evolution of this
process and its contribution to right ventricular (RV) and pulmonary vascular remodeling. We
hypothesize that a detailed characterization of the temporal evolution of vascular
inflammation in PAH and its impact on RV and pulmonary vascular function will add prognostic
value to traditional measures of disease severity and suggest novel therapeutic targets for
future research.

Objectives:

Patients with IPAH and disease-associated PAH will be recruited to the NIH and enrolled in
this natural history study investigating the ability of circulating markers of vascular
inflammation as well as high-resolution cardiac magnetic resonance imaging (MRI) to
accurately stage severity of disease and/or predict clinically relevant outcomes.

Methods:

The total population for the study will be 150 PAH subjects and approximately 55 age and
gender matched controls (i.e. each healthy volunteer is matched to less than or equal to 3
PAH subjects).

PAH subjects will undergo 1) standard clinical examinations including 6-minute walk distance
and echocardiography; 2) cardiopulmonary exercise testing; 3) markers of coagulation and
fibrotic disease; 4) plasma profiling of inflammatory

markers; 5) gene expression profiling of peripheral blood mononuclear cells PBMCs); 6)
high-resolution MRI-based determination of pulmonary vascularand RV structure and function
and 7) Cardiac CT scan.

Plasma markers of endothelial inflammation, PBMC expression profiles, and high-resolution
cardiac MRI will also be studied in age and gender matched controls to define normal ranges
and variability for each of these novel assessments. Comparison of these results to PAH
subjects at baseline will be used to determine the degree to which these investigative tests
distinguish PAH patients from healthy subjects. Likewise, baseline clinical evaluations of
PAH subjects will be used to examine whether any novel test (inflammatory markers, or cardiac
MRI), accurately classifies patients according to their disease severity. In addition, these
tests will be investigated prospectively for their ability to predict PAH disease
progression. Disease progression will be defined prospectively as a decrease in the 6-minute
walk distance of greater than or equal to10% from baseline or clinical worsening requiring an
escalation in therapy, hospitalization due to right heart failure, transplantation or death.

Additional plasma will be collected from PAH subjects and age/gender matched control
subjects. This material will be used to probe for new biomarkers and inflammatory factors
using discovery based approaches (i.e. Proteomics and pulmonary artery endothelial cell
bioassay).

- INCLUSION AND EXCLUSION CRITERIA FOR PAH SUBJECTS

Inclusion Criteria for PAH Subjects:

The following parameters on RHC are required to meet the hemodynamic definition of PAH
(NYHA/WHO Group I PH):

- mean pulmonary artery pressure of greater than 25 mmHg at rest,

- pulmonary capillary wedge pressure of less than or equal to 15 mmHg (or a left
ventricular end-diastolic pressure of less than or equal to 12mmHg) and

- pulmonary vascular resistance of greater than 3 Wood units (240 dyn s cm(5)).

For patients with suspected PAH (Group I PH) who have not undergone a RHC and/or additional
testing to confirm the diagnosis, this testing will be completed as clinically indicated
under a procedural consent. If clinically indicated (diagnostic) testing indicates that the
subject with suspected PAH does not in fact meet standard criteria for PAH (Group I PH),
then the subject will be removed from the study.

Exclusion Criteria for PAH Subjects:

- Pregnant or breastfeeding women (all women of childbearing potential will be required
to have a screening urine or blood pregnancy test)

- Age less than 18 years

- Inability to provide informed written consent for participation in the study

INCLUSION AND EXCLUSION CRITERIA FOR HEALTHY CONTROL SUBJECTS

Inclusion Criteria for Control Subjects

Any healthy man or woman who is the appropriate age and gender for matching to a PAH
patient

- Must be eligible for MRI and Gadolinium Based MRI studies

- Must be eligible for CT and Iodine Based Contrast CT studies

Exclusion Criteria for Healthy Control Subjects

- Current pregnancy or breastfeeding (All women of childbearing potential will be
required to have a screening urine or blood pregnancy test)

- Electrocardiographic evidence of clinically relevant heart disease

- Symptoms of coronary or cardiac insufficiency

- More than one major risk factor for coronary artery disease (excluding age and gender)

- Obesity (defined as a body mass index > 30 kg/m(2))

- History of underlying conditions/risk factors associated with pulmonary hypertension
such as collagen vascular disease, HIV infection, use of appetite suppressants,
chronic liver disease or cirrhosis of the liver, chronic thromboembolic disease,
congenital heart defects, hypoxemia and/or significant pulmonary parenchymal disease

- Systemic hypertension that is not well controlled (i.e. blood pressure at the time of
screening greater than or equal to140/90 mmHg) in adults < 60 years old or greater
than or equal to 150/90 mmHg in adults 60 years or older) on medications. Subjects
taking > 2 anti-hypertensive medications will be excluded irrespective of their
current blood pressure at time of screening

- Anemia, thrombocytopenia or coagulopathy

- Renal insufficiency (defined as an estimated glomerular filtration rate of < 60
mL/min/1.73m(2) of body surface area)

- Active tobacco use (> 6 months) in the past ten years, any tobacco use within 3 months
prior to the screening evaluation or any tobacco use prior to completion of the study

- Inability to provide informed written consent for participation in the study

- History of recreational drug use with the exception of marijuana (as long as marijuana
use was > 3 months from the time of study screening).

Exclusion Criteria for MRI in Healthy Control Subjects and Subjects with PAH

These contraindications include but are not limited to the following devices or conditions:

1. Implanted cardiac pacemaker or defibrillator

2. Cochlear Implants

3. Ocular foreign body (e.g. metal shavings)

4. Embedded shrapnel fragments

5. Central nervous system aneurysm clips

6. Implanted neural stimulator

7. Any implanted device that is incompatible with MRI

8. Unsatisfactory performance status as judged by the referring physician such that the
subject could not tolerate an MRI scan. Examples of medical conditions that would not
be accepted would include unstable angina and severe dyspnea at rest

9. Subjects requiring monitored sedation for MRI studies

10. Subjects with a condition precluding entry into the scanner (e.g. morbid obesity,
claustrophobia, etc.)

11. Subjects with severe back-pain or motion disorders who will be unable to tolerate
supine positioning within the MRI scanner and hold still for the duration of the
examination.

Exclusion Criteria for Gadolinium Based MRI Studies Only:

1. History of severe allergic reaction to gadolinium contrast agents despite pre-
medication with diphenhydramine and prednisone

2. Chronic kidney disease (an estimated glomerular filtration rate of < 60
mL/min/1.73m(2) of body surface area)

Exclusion Criteria for Cardiac Computed Tomography in Healthy Control Subjects and Subjects
with PAH:

1) Subjects with a condition precluding entry into the scanner (e.g. morbid

obesity, claustrophobia, etc.)

Exclusion Criteria for Iodine Based Contrast CTA Studies Only:

1. Serum creatinine > 1.4 mg/dL

2. History of multiple myeloma

3. Use of metformin-containing products less than 24 hours prior to contrast
administration

4. History of significant allergic reaction to CTA contrast agents despite premedication
with diphenhydramine and prednisone