Endothelial Cell Dysfunction in Pulmonary Hypertension

Introduction: Primary pulmonary hypertension, now known as idiopathic pulmonary arterial
hypertension (IPAH), a subgroup of pulmonary arterial hypertension (PAH), is a rare disorder
characterized by severe morbidity and high mortality rates. There are no routine screening
tests or validated markers of disease activity in IPAH, or the broader group of PAH.
Therefore, patients usually present at advanced stages of disease. The pathogenesis of IPAH
and other forms of PAH remain unclear. Prior theories stressed a "one-hit" hypothesis.
Current thinking focuses on a "two-hit" hypothesis: 1) genetic susceptibility, and 2) a
triggering stimulus that initiates pulmonary vascular injury, resulting in endothelial cell
(EC) dysfunction and the mobilization of endothelial progenitor cells (EPC). Loss of function
mutations in the bone morphogenetic protein receptor 2 (BMPR2) gene, has been implicated in
the pathogenesis of IPAH. EC dysfunction in IPAH has been associated with decreases in both
endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) production.
Peripheral blood mononuclear cells (PBMCs) interact with an altered endothelial cell surface,
which may also be important in the disease process.

Objectives: We plan to define a subset of differentially regulated biomarkers in IPAH and PAH
that may lead to earlier diagnosis and better methods for measuring responses to therapy.
Specifically, we hope to identify biomarkers of IPAH and other forms of PAH that is
suggestive of NO therapeutic response and which may be useful in titrating NO therapy. We
also hope to identify novel targets for the development of new therapeutic strategies.

Methods: This study will consist of a pilot study and a primary study. The pilot study will
enroll up to 30 patients and 30 controls in order to obtain completed studies on 10 normal
subjects and 10 patients with PAH. The goal of the pilot study is to determine the best
technique for circulating endothelial cell (CEC) and PBMC identification, quantification, and
isolation and EPC identification and quantification. The subjects in the pilot phase undergo
right heart catheterization to obtain hemodynamics and pulmonary artery blood. Pulmonary
artery and peripheral blood will be used for EPC quantification and CEC and PBMC isolation.
CECs and PBMCs will be studied in depth using high density oligonucleotide microarrays. In
addition, plasma obtained from PAH patients and healthy volunteers will be applied in vitro
to various cell populations suspected to be central to disease pathogenesis including but not
limited to ECs, circulating mononuclear cells, cardiac myocytes and/or vascular smooth muscle
cells. Phenotypic alterations induced by plasma exposure will be assessed using in vitro
assays.

The primary study will recruit the following subject groups: 1) patients with IPAH and other
forms of PAH (vascular injury-induced pulmonary hypertension) who currently are on no
therapy, less than or equal to 6 months of IV therapy, or less than or equal to one year of
oral therapy, 2) patients with pulmonary hypertension (PH) ascribed to a nonvascular injury
process and 3) normal individuals. The following baseline studies will be performed in all
groups: 1) noninvasive assessment of right ventricular (RV) function by echocardiogram and
magnetic resonance imaging (MRI), 2) determination of exercise capacity by cardiopulmonary
stress test and six minute walk, 3) measurement of hemodynamic parameters by right heart
catheterization and 4) characterization of disease phenotype by cell surface markers,
oligonucleotide microarrays, and proteomics using peripheral and pulmonary arterial blood.
EPCs will be quantitated and CECs and PBMCs will be isolated and analyzed by flow cytometry
for expression of cell surface markers involved in coagulation, adhesion, and angiogenesis,
as these are important processes in IPAH and PAH. Furthermore, ECs (identified by positive
and negative selection and isolated by cell sorting) and PBMCs will be studied in depth using
high density oligonucleotide microarrays to more fully characterize their transcriptome.

A major impediment to the widespread use of chronic home based inhaled NO are related to its
delivery system and duration of effects. In PAH patients we plan to study a novel NO delivery
system (INO pulse delivery device). Patients with PAH will be given inhaled NO (20 and 40
ppm) and then placed on inhaled NO using the INO pulse delivery device for 24 hours.
Hemodynamics will be obtained serially with each dose and upon completion of 24 hours of
therapy, pulmonary artery and peripheral blood will be drawn and reexamined by flow
cytometry, microarrays, and proteomics.

We also plan to follow response to standard therapy (as determined by the referring
physician). After the initial day 0 studies, we will restudy the same parameters (excluding
NO studies) in patients with PAH at approximately 4 months, and yearly for 5 years after
therapeutic intervention.

- ELIGIBILITY CRITERIA:

Pilot: The pilot study will enroll two groups of individuals: 1) patients who have either
IPAH or a secondary form known to have similar histopathology (PAH), and 2) age, gender,
and race matched control subjects for each patient.

Main: The main study will enroll three groups of individuals: 1) patients who have either
IPAH or a secondary form known to have similar histopathology (PAH), 2) patients with PH
ascribed to a nonvascular injury process, and 3) age, gender, and race matched control
subjects for each PAH patient. Subjects must be at least 18 years of age and must be able
to provide informed, written consent for participation in this study. There is no exclusion
based on race or gender.

INCLUSION CRITERIA FOR PULMONARY ARTERIAL HYPERTENSION PATIENTS:

The inclusion criteria for this study are as follows:

1. Patients diagnosed with IPAH

2. Patients diagnosed with secondary pulmonary hypertension known to have histopathology
similar to the primary form or PAH. Clinical conditions causing pulmonary hypertension
with histopathology similar to the primary form are listed below.

i. Eisenmenger Syndrome

ii. Collagen vascular disease

iii. Liver disease with portal hypertension

iv. Toxin induced injury (anorexic agents, rapeseed oil)

v. HIV disease

vi. Sickle cell disease

EXCLUSION CRITERIA FOR PATIENTS WITH PULMONARY ARTERIAL HYPERTENSION:

1. Pregnant women (all women of childbearing age will be required to have a screening
urine or blood pregnancy test)

2. Age less than 18 years

3. Inability to provide informed written consent for participation in the study

4. Mean PA less than or equal to 25mmHg or PVR less than 3 wood units

5. PCW greater than 16 mmHg unless increase accounted for by increased transpulmonary
gradient greater than or equal to 10 mmHg

6. Patients receiving more than 1 year of oral therapy or more than 6 months of IV
therapy.

INCLUSION CRITERIA FOR PATIENTS WITH NONVASCULAR INJURY-INDUCED PULMONARY HYPERTENSION:

The inclusion criteria are as follows:

Patients diagnosed with pulmonary hypertension not known to have histopathology similar to
the primary form. Etiologies are listed below.

1. Congenital or acquired valvular or myocardial disease

2. Pulmonary parasitic diseases

3. Arterial hypoxemia with hypercapnea

4. COPD with hypoxemia and forced expiratory volume/forced vital capacity (FEV1/FVC)
greater than 2 standard deviations from normal

5. Interstitial lung disease with reduced total lung capacity greater than 2 standard
deviations from normal and infiltrates on chest x-ray

6. Pulmonary thromboembolic disease as evidenced by lung perfusion scan or pulmonary
angiogram, or intravenous drug abuse

7. Pulmonary hypertension due to congenital abnormalities of the lungs, thorax and
diaphragm

EXCLUSION CRITERIA FOR PATIENTS WITH NONVASCULAR INJURY INDUCED PULMONARY HYPERTENSION:

1. Pregnant women (all women of childbearing age will be required to have a screening
urine or blood pregnancy test)

2. Age less than 18 years

3. Inability to provide informed written consent for participation in the study

4. Mean PA less than or equal to 25mmHg or PVR less than 3 wood units

EXCLUSION CRITERIA FOR MRI IN SUBJECTS WITH PULMONARY HYPERTENSION:

1) Implanted cardiac pacemaker or defibrillator

2) Central nervous system aneurysm clips

3) Cochlear implants

4) Neural stimulator

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

6) Insulin pump

7) Metal shrapnel or bullets

8) Claustrophobia.

Furthermore, the following patient groups will be excluded from studies involving the
administration of MRI contrast agents:

1) lactating women

2) renal disease (CrCl less than 20 ml/min)

The creatinine clearance (CrCl) will be calculated using the Cockroft formula where age is
in years, kg is weight in kilograms, and Cr is the serum creatinine. If there is no history
of kidney disease from the patient or referring physician, additional testing will not be
performed. If a patient has a history of renal insufficiency, a recent blood Cr will be
used unless the physician performing the test believes the Cr may have changed since the
last test. If the Cr may have changed, a blood sample will be obtained for Cr or the
subject will be excluded from receiving gadolinium.

CrCl = (140-age) (wt in kg)/72 X serum Cr (mg/dl) for men

CrCl = (0.85) (140-age) (kg)/72 X serum Cr (mg/dl) for women

INCLUSION CRITERIA FOR CONTROL SUBJECTS:

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

2. No history of HIV infection

3. EKG and echocardiogram with no evidence of clinically relevant heart disease

4. Spirometry with no evidence of clinically relevant lung disease

5. No history of causes of pulmonary hypertension such as collagen vascular disease,
chronic liver disease with ALT or AST greater than 2 times the upper limit of normal
or cirrhosis of the liver, chronic thromboembolic disease, congenital heart defects,
or pulmonary parenchymal disease with hypoxemia

6. No history of diseases thought to be related to development of endothelial dysfunction
including systemic hypertension or diabetes requiring drug therapy,
hypercholesterolemia and obesity

7. No history of anemia, thrombocytopenia or coagulopathy

8. No history of renal insufficiency

9. No medical conditions requiring chronic medication use with the exception of:

a. Heartburn, GERD

b. Environmental allergies, post nasal drip or non-allergic rhinitis

c. Asthma with no history of oral steroid use, weekly inhaled steroids, or
hospitalization for asthma exacerbation

d. Dermatologic conditions that do not require the use of oral steroids or other
immunosuppressants

e. Treated and stable thyroid disease, depression, or anxiety.

10. No more than 20 cigarettes per years for the previous 2 years and no cigarette use for
30 days prior to the screening evaluation until completion of the study

EXCLUSION CRITERIA FOR CONTROL SUBJECTS:

1. Current pregnancy, lactation or women not currently using medically acceptable birth
control. (All women of childbearing age will be required to have a screening urine or
blood pregnancy test)

2. Contraindication to MRI scanning including individuals with the following devices:

A) Implanted cardiac pacemaker or defibrillator

B) Central nervous system aneurysm clips

C) Cochlear implants

D) Implanted Neural stimulator

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

F) Insulin pump

G) Metal shrapnel or bullet

H) Claustophobia

3. Contraindications to MRI contrast agent administration

4. Inability to provide informed written consent for participation in the study

5. Chronic, medically refractory atrial tacharrhythmias

6. Symptoms of heart failure.

7. Mean PA greater than 25mmHG or PVR greater than 1.5 wood units, or PCWP greater than
15 mmHg

8. History of recreational drug use with the exception of marijuana. No marijuana use
within 3 months of protocol screening through completion of the study.

9. Intravenous drug abuse.

Volunteers may be excluded if in the opinion of the study investigators they have a
condition that may adversely affect the outcome of the study or the safety of the
volunteer.