Grantee Research Project Results

2008 Progress Report: Human Clinical Studies of Concentrated Ambient Ultrafine and Fine Particles

EPA Grant Number: R832415C003
Subproject: this is subproject number 003 , established and managed by the Center Director under grant R832415
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: Rochester PM Center
Center Director: Oberdörster, Günter
Title: Human Clinical Studies of Concentrated Ambient Ultrafine and Fine Particles
Investigators: Frampton, Mark W. , Zareba, Wojciech , Utell, Mark J. , Oakes, David , Phipps, Richard , Gelein, Robert
Current Investigators: Frampton, Mark W. , Utell, Mark J. , Zareba, Wojciech , Phipps, Richard , Gelein, Robert , Oakes, David
Institution: University of Rochester
EPA Project Officer: Chung, Serena
Project Period: October 1, 2005 through September 30, 2010 (Extended to September 30, 2012)
Project Period Covered by this Report: October 1, 2007 through September 30,2008
RFA: Particulate Matter Research Centers (2004) RFA Text | Recipients Lists
Research Category: Human Health , Air

Objective:

The overall objective of our current and planned studies is to determine the pulmonary and cardiovascular effects of exposure to ultrafine and fine particulate matter (PM). The clinical studies in healthy humans and susceptible individuals with diabetes proposed in this research core focus on the effects of ambient ultrafine and fine particles on three major determinants of adverse cardiac events: 1) blood coagulation induced by effects on platelets and circulating microparticles; 2) cardiac output; and 3) cardiac rhythm and repolarization.

Our overall hypothesis is that inhalation of ambient PM causes small but measurable changes in coagulation and cardiovascular function that help explain the cardiovascular effects of PM exposure. We further hypothesize that the cardiovascular effects are determined by the ability of PM to generate reactive oxygen and nitrogen species, and are more pronounced in subjects with type 2 diabetes. Inhaled ultrafine particles increase the burden of reactive oxygen species to the endothelium. Endothelial activation and vasoconstriction increase platelet adherence and release of thromboxane, activate and prolong the transit time of blood leukocytes, and deplete vascular nitric oxide (NO). Particles may also have direct effects on platelets and leukocytes. Vascular injury triggers release of procoagulant microparticles into the blood, and initiation of coagulation. In collaboration with the Vascular and Inflammation Facility Core, we measure the effects of inhaled ambient fine PM on platelet number, phenotype, and function, and quantitate intravascular microparticles derived from platelets and endothelial cells. In collaboration with the Cardiac Core, we use noninvasive monitoring methods to measure exposure effects on cardiac output, rhythm, and repolarization. These studies take advantage of and extend upon our project funded by the National Institute of Environmental Health Sciences and the EPA, “Ultrafine Particle-Induced Oxidative Stress”, which focuses on effects on vascular function and NO.

In Rochester, New York 75 patients taking part in a cardiac rehabilitation program will be studied. Patients from an active cardiac rehabilitation program within the University of Rochester Medical Center are offered enrollment in the health effects study as they enter the Cardiac Rehabilitation program. These are patients who have had a recent coronary event such as myocardial infarction or unstable angina leading to coronary stenting. The program involves supervised, graded twice or thrice weekly exercise sessions for a total of 10 weeks. As part of the rehabilitation protocol, vital signs and a standard 12 lead EKG will be performed. In addition to the regularly electrophysiologically monitored exercise of the rehabilitation program, subjects will undergo continuously recorded Holter ECG recordings performed and analyzed by the Cardiac Core allowing evaluation of a series of ECG parameters at rest, during exercise, and during immediate post-exercise period. Venous blood samples will be obtained once per week and analyzed by the Vascular & Inflammation Core for acute phase reactants (fibrinogen and C-reactive protein) previously found to vary with ultrafine particle exposure, as well as complete blood counts. Concurrently, ultrafine particle number and particle mass will be measured continuously at a central measuring site in downtown Rochester. Other EPA Criteria Pollutants are also measured in eastern Rochester as well. In addition, one-third of the patients are being asked to do personal particle count monitoring in their car to and from the rehab facility and in their homes for 48 hours using a portable nuclei counter (TSI model 3781). Levels of ambient ultrafine and fine particles will then be associated with health data from the cardiac rehabilitation panel study.

Approach:

Three human exposure protocols will be conducted using the Harvard ultrafine ambient particle concentrator, in collaboration with the Aerosol Generation and Analysis Facility Core. The first protocol will examine effects in healthy subjects, the second protocol will examine effects in age-matched subjects with type 2 diabetes, and the third protocol will assess the role of pretreatment with the cyclooxygenase inhibitor aspirin in preventing the cardiovascular effects of ultrafine/fine particle exposure. In collaboration with the Vascular and Inflammation Facility Core, we will determine particle effects on platelet function and release of endothelial and platelet microparticles into the circulation, and will examine effects on platelet-leukocyte adhesion, bone marrow stimulation, and changes in gene expression in blood mononuclear cells. Continuous ECG monitoring, in collaboration with the Cardiac Facility Core, will detect changes in cardiac repolarization, and noninvasive impedence cardiography will measure changes in cardiac output. Genomic DNA from exposed subjects will be analyzed for candidate gene polymorphisms identified in Research Core #2, Epidemiological Studies. Exposure studies will be designed and conducted in parallel with similar animal exposure studies conducted by Research Core #4, Animal Models. The impact of PM-associated reactive oxygen species, size, composition, and source will be examined in collaboration with Research Core #1, Characterization and Source Apportionment, and with the Biostatistics Facility Core.

Progress Summary:

Human clinical exposures to concentrated ambient ultrafine particles The Harvard ultrafine particle concentrator has been installed in a dedicated room in the Kornberg Medical Research Building at the University of Rochester Medical Center, and is fully operational. We have completed construction of a negative-pressure inhalation chamber, which is housed within our new exposure facility. We have initiated human clinical exposures to concentrated ambient ultrafine particles, using the concentrator and the exposure chamber. Installation and maintenance of the concentrator and exposure system requires close collaboration with Dr. Oberdörster, Research Core #1, and the Aerosol Generation & Analysis Core.

We have now completed a clinical study of healthy never-smoking subjects inhaling concentrated ambient UFP using the concentrator. This study is co-funded by an NIEHS RO1 grant and this EPA center grant. Subjects are 20 healthy never-smokers, age 30 to 60 yrs, stratified by age and gender. Subjects are admitted to the Clinical Research Center the day prior to exposure, to minimize the potential influence of outdoor pollutant exposure in the hours prior to the experimental exposure. Physiologic measurements are made the day prior to exposure, and then 0, 3.5, 21, and 45 hours after exposure. Measurements include blood sampling for flow cytometry and soluble markers of inflammation and coagulation, pulmonary function testing, diffusing capacity for carbon monoxide, and flow-mediated dilatation of the forearm. The EPA Center is funding continuous cardiac monitoring, noninvasive measurement of cardiac output, and newly developed methods for analysis of platelet activation and circulating microparticles, using flow cytometry.

All 20 subjects have completed both exposures. Exposures have been successful from a technical standpoint, and no subject has experienced symptoms or problems with the exposures. Data analysis is underway. Figure 1 shows the mean (SD) concentrated particle number concentrations for each subject’s UFP exposure.

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There was considerable variability in exposure concentrations, as expected. Preliminary findings suggest UFP exposure resulted in small increases in diastolic and mean blood pressure immediately after exposure (Figure 2), without changes in heart rate.

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There were small reductions in the FEV1 and mid-expiratory flow rates after UFP exposure relative to air (Figure 3). Flow cytometry analyses, measurement of markers of endothelial and vascular effects, and ECG analyses are underway.

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2. Inhalation of carbon UFP in diabetics

We have completed our study of the effects of inhalation of ultrafine carbon particles in subjects with diabetes. Diabetics have vascular endothelial dysfunction which may increase their risk for adverse cardiovascular effects from airborne particles. Type 2 diabetics, age 30-60, without clinical cardiovascular disease and not on “statin” medications, were exposed to filtered air or 50 μg/m3 carbon UFP (count median diameter ~30 nm, GSD 1.8) by mouthpiece for two hours, in a randomized double-blind cross-over study. Exposures were separated by at least two weeks. Nineteen subjects completed the study.

A preliminary analysis of the findings was published in abstract form and presented at the 2007 American Thoracic Society International Conference. Compared with air exposure, UFP exposure increased platelet expression of CD40 ligand (CD40L) (Figure 4), a marker of platelet activation and a key molecule in the development of atherosclerosis.

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UFP exposure also increased platelet-associated tissue factor (TF) and increased the number of microparticles expressing TF (Figure 5).

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Changes occurred most consistently 3.5 hours after exposure, and effects were no longer seen 24 hours after exposure. There were no significant effects of UFP on platelet counts or platelet aggregates. The subjects with diabetes showed significant reductions in forearm flowmediated vascular dilatation in comparison with healthy subjects, as expected. Both the pulmonary diffusing capacity for carbon monoxide (an indicator of pulmonary vascular function), and forearm flow mediated dilatation (an indicator of systemic vascular function), decreased with UFP exposure to ultrafine particles compared with clean air exposure, but the differences were not statistically significant.

Analysis of continuous ECG recordings suggests delayed effects of UFP exposure on heart rate. Analysis of the interval between normal beats (NN interval), which is inversely related to heart rate, showed a decrease over time relative to air exposure. When heart rate was averaged over the duration of each multi-hour ECG recording interval, heart rate increased during the 48 hrs after UFP, relative to air exposure (Figure 6).

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We concluded that inhalation of carbon UFP for 2 hours may transiently activate vascular endothelium and/or platelets in subjects with type 2 diabetes. This finding supports the hypothesis that exposure to ambient UFP may increase the potential for vascular thrombosis in patients with severe vascular disease or ulcerated atherosclerotic plaques. The reasons for the delayed increase in heart rate are unclear, but increased heart rate increases the oxygen demand of the heart, and could adversely affect people with angina or coronary artery disease. We are currently working closely with the Biostatistics Core, using a 2-model analysis to examine effects of gender and age on the findings. This project also involved close collaboration with Research Core #1 and the Aerosol Generation & Analysis Core for UFP generation and exposure monitoring. Manuscript preparation is underway.

3. Ultrafine Particles Activate Platelets In Vitro

Inhaled UFP have the capability of entering pulmonary vascular endothelial cells and even the blood. One possible mechanism for activation of platelets, thus increasing risk for cardiovascular thrombotic events, may involve direct effects of UFP that have entered the circulation. The purpose of this study is to examine the effects of both laboratory-generated carbon ultrafine particles, and ambient UFP, on platelet activation in vitro. The following particles (and count diameters) were studied: copper (0.022 μm), commercial elemental carbon (P90, 0.014 μm), laboratory-generated elemental carbon (lab carbon, 0.030 μm), and diesel exhaust particles (DEP, 1.62 μm). Vortexed particle suspensions were added to whole blood from seven healthy never-smoking subjects and incubated for 30 minutes at 37ºC. Platelet expression of CD62P (p-selectin), platelet aggregates, and platelet-leukocyte conjugates were measured by flow cytometry. Dose-dependent increases in the expression of CD62P were most pronounced with copper and lab carbon. Lab carbon also significantly increased platelet aggregates and platelet-leukocyte conjugates. These findings indicate that UFP can activate platelets in vitro, and the activity varies with particle size and composition.

We have further examined effects of lab carbon UFP on platelet activation and conjugates in the presence of low concentrations of platelet agonists. Carbon UFP at 20 μg/ml significantly increased platelet p-selectin expression (p = 0.04), but did not further enhance pselectin expression in the presence of platelet agonists. There were no significant increases in platelet aggregates or microparticles. Carbon UFP at 20 μg/ml also significantly reduced the platelet count in the presence of all agonists except thrombin, suggesting that low concentrations of agonists enhanced the formation of platelet-leukocyte aggregates.

These studies suggest that UFP, to the degree that they gain access to pulmonary capillary blood following inhalation, have the potential to directly activate platelets and enhance the formation of leukocyte-platelet aggregates in the presence of low concentrations of platelet aggregates. This provides a further mechanism for the acute cardiovascular effects of PM exposure.

To date, 40 of the 75 patients have been enrolled. Thirty-six patients have completed the entire protocol; the patients include 22 men and 9 women; mean age = 64 years and range = 38 to 80 years)). Four patients are in various stages of the 10-week study. Five patients withdrew during the study for various reasons. We are actively screening several potential patients. To make the ambient measurements of ultrafine particles relevant to subject exposures, we are enrolling subjects who live within 5 miles of the central monitoring site or the cardiac rehab center. Ultrafine particle exposures within and without the cardiac rehab center are being monitored continuously.

In order to meet the inclusion criteria, participants must have stable coronary artery disease, must be enrolled in the University of Rochester Medical Center rehabilitation program, and must live in the Rochester area. Participants have to be current non-smokers and should be in the study area during the entire study period. Participants have to be able too physically and mentally comply with the cardiac rehabilitation protocol. Patients with atrial fibrillation, pacemakers, bundle-branch blocks, type 1 diabetes and patients that are away from the study area for an extended period of time will not be included in the study. Annual approval for the protocol has been received from the University of Rochester Research Subjects Review Board (RSRB).

Particle Monitoring Intermediate Results:

Measurements of ultrafine particles are proceeding as proposed with continuous monitoring of ambient particles within and outside of the cardiac rehab center. The particle size distribution data from the Cardiac Rehabilitation Center (indoors and outdoors) and at the NYS DEC site are being collected to support the clinical studies of heart rate variability and inflammatory markers in the blood of rehabilitation patients. Fifteen patients have completed the take home monitoring, with 11 of those doing automobile monitoring.

Monitoring period for September 2006 through December 2007. 94% ultrafine particle monitoring rate was recorded at the rehab facility. 82% ultrafine particle monitoring rate was recorded at the DEC site. Average commute was 15 min. each way for the 11 patients with monitoring performed in their automobile.

Hourly Ultrafine Particle Count Concentrations (x103 p/cm3) (Sept. 06 – Dec.

07)

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Hourly DEC Data (September 2006 – December 2007)

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We are examining the relationship between the ambient ultrafine particle size distributions at the Rehabilitation Facility and the measurements at the NYSDEC site during a one-year period. The data have been collected and the analyses are underway. The goal is to examine the differences in ultrafine particle counts in 2 areas that are closely located.

Analytical Approach:

In our analytical plan, we will explore the relationship between closeness of home to roadway and traffic and clinical responses to ultrafine particles in the ongoing panel study. At present, we have zip codes and addresses of all patients enrolled in the study. We have identified the outcome variables and what the outcome variables will be adjusted for.

Currently we are exploring several models for the analysis.

1) General Strategy: We are developing the analytical plan with assistance from our biostatistical core. A key strength of this study is the availability of longitudinal measurements on each subject, corresponding to their successive visits to the rehabilitation center. This design feature allows each subject to be used as his or her own control. Differences between subjects may be eliminated by stratification, corresponding to the use of a fixed effects analysis of variance model, or by explicitly modeling subject effects as random. The latter approach is necessary for examining the influence of factors that do not change from one visit to another, for example the gender of the subject. The former approach will give greater statistical power to address the primary hypotheses of the study relating to the influence of the UFP concentrations, which do change from visit to visit. In the simplest case, the fixed approach corresponds to a simple paired t-test, comparing responses of the same subject during periods of relatively high and low exposure to ultrafine particles. More commonly analysis of covariance (PROC GLM) will be used, with the subjects entered as fixed effects and ultrafine particle exposure as a continuous variable. Models with subjects entered as random effects will be used to assess the generalizability of the findings to other populations. The random effects approach requires specification of the correlation structure of the data. All proposed analyses can be implemented in SAS using PROC MIXED and PROC GENMOD.

2) Multivariable Analysis: In view of the complex relationship between UFP levels and weather (which may independently influence cardiac responses) it will be necessary to examine multivariable models including effects of variations in temperature and relative humidity as well as calendar (e.g. day of week) effects. The functional forms of the potential confounding effects will be explored using penalized spline methodology.

3) Sample size: The proposed net sample size of 75 subjects will allow detection of an effect size of 33% (ratio of mean difference to standard deviation. of difference) in a response variable with 80% power, using a two-sided test with alpha level = 0.05.

4) Progress to date: Data on cardiac responses including perceived exertion, changes in heart rate from pre-exercise to peak and to post exercise, associated changes in systolic and diastolic blood pressure, and in white blood count has been examined on the first 12 subjects to complete the program. Correlations have been assessed with measures of ultrafine particle exposure at the corresponding visit. The primary purpose of performing these analyses has been to check the data acquisition and management process, which requires merging of data from several sources. In the course of performing these analyses we have identified a number of data issues which have now been resolved.

Expected Results:

Confirmation of our hypothesis that exposure to ambient ultrafine and fine particles promotes coagulation and alters cardiac function will have important implications for air pollution regulatory efforts, and will provide new approaches for the prevention of cardiovascular health effects.

Future Activities:

We are in the planning and approval stages of our next clinical protocol, examining concentrated UFP effects in people with type 2 diabetes. This study will provide a comparison with our just-completed study in healthy subjects, and with our findings in diabetics exposed to elemental carbon UFP.

We have applied for and received pilot study funds from the EPA Center to examine the effects of ambient ultrafine particles on blood dendritic cells (DC). The DC is a key controller of the allergic immune response, by processing inhaled allergens and foreign proteins and presenting antigens to T lymphocytes. We hypothesize that inhalation of ambient UFP contributes to the worsening of airways disease in people with asthma by altering DC maturation and function, driving the immune response to a Th2-type, or allergic, phenotype. In the last five subjects of our UPCON study, we have obtained blood before and 24 hours after exposure, begun to analyze peripheral blood DC phenotype and function, with the help of Drs. Stephen Georas and Marc Williams of the Pulmonary and Critical Care Division. We have submitted a protocol to the Research Subjects Review Board, and to the EPA, to study subjects with mild to moderate asthma. These subjects will inhale air or concentrated ambient UFP for two hours, and blood dendritic cells will be analyzed and compared with the findings in healthy subjects. In addition, lung function and exhaled breath markers of inflammation will determine whether dendritic cell effects correlate with airway effects. This pilot study is planned as the first effort in a new direction for the clinical core of the Center, examining the mechanisms for airway effects of PM in asthma.

For the in vitro studies of platelet activation, future experiments will explore the effects of ambient UFP collected from different sites, additional types of diesel exhaust, as well as the mechanisms for particle activation of platelets. We are collaborating with Dr. Phipps and the Vascular & Inflammation Core in developing these research strategies.

We will continue patient recruitment into the protocol. At the present time, our recruitment rates are moving ahead according to the proposed time line; we have completed enrollment of more than 50% of the proposed population into the study. We will continue particle measurements in homes and automobiles with a plan to sample these sites in approximately 25% of the enrolled population.

Journal Articles:

No journal articles submitted with this report: View all 57 publications for this subproject

Supplemental Keywords:

ultrafine particles; endothelial dysfunction; air pollution, cardiovascular health,, RFA, Health, PHYSICAL ASPECTS, Scientific Discipline, Air, particulate matter, Health Risk Assessment, Risk Assessments, Physical Processes, atmospheric particulate matter, acute cardiovascular effects, long term exposure, atmospheric particles, airway disease, exposure, ambient particle health effects, human exposure, ultrafine particulate matter, atmospheric aerosol particles, PM, aersol particles, cardiovascular disease

Progress and Final Reports:

Original Abstract

Main Center Abstract and Reports:

R832415 Rochester PM Center

Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R832415C001 Characterization and Source Apportionment
R832415C002 Epidemiological Studies on Extra Pulmonary Effects of Fresh and Aged Urban Aerosols from Different Sources
R832415C003 Human Clinical Studies of Concentrated Ambient Ultrafine and Fine Particles
R832415C004 Animal models: Cardiovascular Disease, CNS Injury and Ultrafine Particle Biokinetics
R832415C005 Ultrafine Particle Cell Interactions In Vitro: Molecular Mechanisms Leading To Altered Gene Expression in Relation to Particle Composition

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