Cardiovascular Effects of Exposure to Ozone

This multicenter study will investigate whether short-term exposure of elderly volunteers to
ambient levels of O3 in a controlled exposure setting while intermittently exercising causes
acute cardiovascular responses. The study is based on the suppositions that: 1) elderly
people are a susceptible group for cardiovascular effects; and 2) effects are more likely
with exercise.

The study will involve approximately 90 healthy volunteers aged ≥55 and ≤70 who meet strict
criteria for inclusion. They will be exposed for 3 hours to clean air, 0.07 ppm O3 (near the
current NAAQS), and 0.12 ppm O3 (a level measured in several locations in the US). A suite
of cardiovascular and pulmonary endpoints will be measured on the day before the exposure
and up to 22 hours after the exposures. The study is being conducted at three centers using
a common protocol and common SOPs. Most the endpoints will be analyzed by core laboratories
to reduce the variability in the results. A Data Coordination and Analysis Center will
assemble all the data generated by the three centers and conduct the statistical analyses on
the combined data sets.

The study has 3 main objectives:

1. To determine the relationship of altered autonomic balance (measured as changes in
heart rate and heart rate variability (HRV)), cardiac arrhythmia, and repolarization
and ozone exposure.

2. To identify instances of altered systemic vascular function [measured as brachial
artery flow-mediated dilation (FMD)without and with nitroglycerin (NTG) when exposed to
ozone.

3. To identify pro-thrombotic vascular state (measured as increase in von Willebrand
factor antigen in blood - primary endpoints) when exposed to ozone.

Additional objectives include:

1. To identify any increase in micro particle-associated tissues factor (measured as
number of particles and tissue factor activity) and platelet activation) in ozone
exposure.

2. To identify if markers of systemic oxidative stress and inflammation and any
correlation with the cardiovascular effects and degree of airway injury (measured as
CC16) and airway inflammatory effects (neutrophils and cytokines in induced sputum) in
ozone exposure.

3. To determine if cardiovascular effects in ozone exposure are correlated with airway
inflammatory effects, but not lung function effects.

Inclusion Criteria:

- males and females of all ethnic backgrounds.

- Normal spirometry (FEV1 and FVC >75% of predicted and FEV1/FVC >0.65).

- Ability to complete the exposure exercise regimen chosen to induce a ventilation rate
of 15 to 17 L/min/m2 without exceeding 80% of predicted maximal heart rate.

- Normal baseline 12-lead resting ECG, and absence of significant ST depression while
performing the 15-minute required level of exercise targeted for the exposure period.

- Subjects must be able to avoid certain medication supplements listed for 1 week
before the exposure.

Exclusion Criteria:

- Non-English speaking.

- Including, but not limited to as ascertained by the physicians: Subjects with chronic
cardiovascular (such as ischemic heart disease) or respiratory (such as asthma or
COPD) disease; diabetes, or other organ or system dysfunction; cerebrovascular
disease; active psychiatric disorders that would interfere with the subject's ability
to understand and participate in the study. Subjects who have tested positive for a
disease that affects the immune system (such as HIV, lymphoma, leukemia) or current
drug or alcohol abuse (defined as having more than 3 drinks per day or being unable
to abstain from alcohol for 3 days).

- Subjects with atopy or allergic rhinitis will not be excluded as long as they do not
require regular treatment with antihistamines or systemic steroids.

- Ever-smokers (smoked tobacco or marijuana during the last five years, or with history
of >10 pack year for tobacco or > 1 joint year for marijuana, or living with a smoker
who smokes inside the house).

- Subject having plasma cotinine level > 3ng/mL.

- BMI >35 or <18 (35 is the official cut off for class 1 obesity).

- Hypertension (defined as blood pressure >140 systolic or >90diastolic) or on
anti-hypertension medications other than diuretics.

- Pregnancy or nursing (breastfeeding).

- On the following medications: prednisone, statins, beta-blockers, anticoagulants,
current hormonal therapy, tamoxifen. Subjects will not be asked to discontinue needed
prescription medications for the purpose of this study. If any of these medications
becomes necessary during the course of the study, the subjects will be excluded. Use
of other medications will be considered on an individual basis.

- Subjects taking aspirin or PDE5 inhibitors must be willing to abstain from these
medications during the week preceding each exposure.

- Occupational exposures (exposed to high levels of vapors, dust, gases, or fumes on an
on-going basis)