Air Pollution and the Microvasculature: A Cross-Sectional

Assessment of In Vivo Retinal Images in the PopulationBased Multi-Ethnic Study of Atherosclerosis (MESA)

Sara D. Adar1,2,3*, Ronald Klein4, Barbara E. K. Klein4, Adam A. Szpiro5, Mary Frances Cotch6, Tien Y.

Wong7,8, Marie S. O’Neill3,9, Sandi Shrager8

, R. Graham Barr10, David S. Siscovick1,11, Martha L.

Daviglus12, Paul D. Sampson13, Joel D. Kaufman1,2,11

1 Department of Epidemiology, University of Washington, Seattle, Washington, United States of America, 2Department of Environmental and Occupational Health

Sciences, University of Washington, Seattle, Washington, United States of America, 3 Department of Epidemiology, University of Michigan, Ann Arbor, Michigan, United

States of America, 4 Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, Wisconsin, United States of America, 5Department of

Biostatistics, University of Washington, Seattle, Washington, United States of America, 6 Division of Epidemiology and Clinical Applications, National Eye Institute,

Bethesda, Maryland, United States of America, 7 Department of Ophthalmology, University of Melbourne, Melbourne, Victoria, Australia, 8 Singapore Eye Research

Institute, National University of Singapore, Singapore, 9 Department of Environmental Health Sciences, University of Michigan, Ann Arbor, Michigan, United States of

America, 10 Departments of Medicine and Epidemiology, Columbia University Medical Center, New York, New York, United States of America, 11 Department of Medicine,

University of Washington, Seattle, Washington, United States of America, 12 Department of Preventive Medicine and Medicine, Northwestern University, Chicago, Illinois,

United States of America, 13 Department of Statistics, University of Washington, Seattle, Washington, United States of America

Abstract

Background: Long- and short-term exposures to air pollution, especially fine particulate matter (PM2.5), have been linked to

cardiovascular morbidity and mortality. One hypothesized mechanism for these associations involves microvascular effects.

Retinal photography provides a novel, in vivo approach to examine the association of air pollution with changes in the

human microvasculature.

Methods and Findings: Chronic and acute associations between residential air pollution concentrations and retinal vessel

diameters, expressed as central retinal arteriolar equivalents (CRAE) and central retinal venular equivalents (CRVE), were

examined using digital retinal images taken in Multi-Ethnic Study of Atherosclerosis (MESA) participants between 2002 and

2003. Study participants (46 to 87 years of age) were without clinical cardiovascular disease at the baseline examination

(2000–2002). Long-term outdoor concentrations of PM2.5 were estimated at each participant’s home for the 2 years

preceding the clinical exam using a spatio-temporal model. Short-term concentrations were assigned using outdoor

measurements on the day preceding the clinical exam. Residential proximity to roadways was also used as an indicator of

long-term traffic exposures. All associations were examined using linear regression models adjusted for subject-specific age,

sex, race/ethnicity, education, income, smoking status, alcohol use, physical activity, body mass index, family history of

cardiovascular disease, diabetes status, serum cholesterol, glucose, blood pressure, emphysema, C-reactive protein,

medication use, and fellow vessel diameter. Short-term associations were further controlled for weather and seasonality.

Among the 4,607 participants with complete data, CRAE were found to be narrower among persons residing in regions with

increased long- and short-term levels of PM2.5. These relationships were observed in a joint exposure model with 20.8 mm

(95% confidence interval [CI] 21.1 to 20.5) and 20.4 mm (95% CI 20.8 to 0.1) decreases in CRAE per interquartile increases

in long- (3 mg/m3

) and short-term (9 mg/m3) PM2.5 levels, respectively. These reductions in CRAE are equivalent to 7- and 3-

year increases in age in the same cohort. Similarly, living near a major road was also associated with a 20.7 mm decrease

(95% CI 21.4 to 0.1) in CRAE. Although the chronic association with CRAE was largely influenced by differences in exposure

between cities, this relationship was generally robust to control for city-level covariates and no significant differences were

observed between cities. Wider CRVE were associated with living in areas of higher PM2.5 concentrations, but these findings

were less robust and not supported by the presence of consistent acute associations with PM2.5.

Conclusions: Residing in regions with higher air pollution concentrations and experiencing daily increases in air pollution

were each associated with narrower retinal arteriolar diameters in older individuals. These findings support the hypothesis

that important vascular phenomena are associated with small increases in short-term or long-term air pollution exposures,

even at current exposure levels, and further corroborate reported associations between air pollution and the development

and exacerbation of clinical cardiovascular disease.

Please see later in the article for the Editors’ Summary.

PLoS Medicine | www.plosmedicine.org 1 November 2010 | Volume 7 | Issue 11 | e1000372
Citation: Adar SD, Klein R, Klein BEK, Szpiro AA, Cotch MF, et al. (2010) Air Pollution and the Microvasculature: A Cross-Sectional Assessment of In Vivo Retinal

Images in the Population-Based Multi-Ethnic Study of Atherosclerosis (MESA). PLoS Med 7(11): e1000372. doi:10.1371/journal.pmed.1000372

Academic Editor: Bruce P. Lanphear, Simon Fraser University, Canada

Received January 27, 2010; Accepted October 18, 2010; Published November 30, 2010

This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public

domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.

Funding: This work was supported by awards RD831697 from the Environmental Protection Agency; EY000403 from the National Eye Institute; HL69979, N01-

HC-95159 through N01-HC-95165, and N01-HC-95169 from the National Heart, Lung, Blood Institute; and K24ES013195, P50ES015915, P30ES07033 and

R01ES016932 from the National Institutes of Environmental Health Sciences. The funders had no role in study design, data collection and analysis, decision to

publish, or preparation of the manuscript.

Competing Interests: Several authors (SDA, AAS, MSO, RGB, DSS, PDS, and JDK) have received unrestricted grant funding or support on unrestricted grant

funds to study related topics from the Environmental Protection Agency, National Institutes of Heath, and/or the Health Effects Institute.

Abbreviations: AQS, Air Quality System; BMI, body mass index; CI, confidence interval; CRAE, central retinal arteriolar equivalents; CRVE, central retinal venular

equivalents; df, degrees of freedom; EPA, Environmental Protection Agency; HDL, high-density lipoprotein; LDL, low-density lipoprotein; MESA, Multi-Ethnic Study

of Atherosclerosis; MESA Air, Multi-Ethnic Study of Atherosclerosis and Air Pollution; PM2.5, fine particulate matter less than 2.5 mm in aerodynamic diameter; SD,

standard deviation.

* E-mail: sadar@umich.edu

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Introduction

Long- and short-term exposures to ambient and traffic-related

air pollution, especially fine particulate matter (particulate matter

less than 2.5 mm in aerodynamic diameter, or PM2.5), have been

linked to higher rates of cardiovascular morbidity and mortality

[1–5]. It has been hypothesized that impacts on microvascular

function may play a role in these associations [6]. Toxicological

studies support this hypothesis as they have demonstrated

that PM2.5 can impair microvascular endothelium-dependent

dilation [7]. One human study also demonstrated air pollution

and exercise-induced ischemia in a pattern that was more

consistent with impaired myocardial microvascular flow than

altered larger epicardial vessel coronary circulation [8]. Other

human studies have shown links between air pollution and

impaired vasodilatation or enhanced vasoconstriction in the

forearm [9–12], but none have directly explored associations with

the microvasculature.

Retinal photography provides a noninvasive, in vivo, method

for characterizing the human microvasculature since retinal vessels

are 60–300 mm in diameter. Several studies have found that

narrower arteriolar diameters and wider venular diameters, as

measured by retinal photography, are each associated with

increased risk of myocardial infarction, stroke, and cardiovascular

mortality, independent of other traditional risk factors [13–19].

Furthermore, recent data from the Multi-Ethnic Study of

Atherosclerosis (MESA) have linked narrower retinal arteriolar

diameters with measures of subclinical cardiovascular disease

including increased left ventricular mass, reduced aortic distensibility, and reduced large artery compliance [20–22]. These

findings were independent of other cardiovascular risk factors,

including blood pressure, diabetes, blood cholesterol level, family

history of cardiovascular disease, body mass index (BMI), glucose

level, and C-reactive protein, which have also been associated with

retinal vessel diameter in the same cohort [23]. It is unknown,

however, if exposures to ambient and traffic-related air pollution

are related to adverse retinal microvascular changes

Our primary aim was to investigate cross-sectional associations

between long- and short-term air pollution concentrations and

microvascular characteristics using arteriolar vessel diameter as

measured by retinal photography in MESA, a well-characterized

cohort with individual-level predictions of residential pollutant

concentrations. We hypothesized that higher air pollution levels

would be associated with narrowed retinal arteriole diameters. We

also hypothesized that greater air pollution concentrations would

be associated with widened retinal venular diameters.

Methods

Study Population

MESA is a prospective cohort study designed to investigate the

progression of subclinical and clinical cardiovascular disease [24].

The cohort is comprised of 6,814 white, African American,

Hispanic, and Asian (of Chinese decent) men and women

recruited in six American communities (Baltimore, MD; Chicago,

IL; Forsyth County, NC; Los Angeles County, CA; Northern

Manhattan, NY; and St. Paul, MN). Participants were aged 45 to

84 y and free of clinical cardiovascular disease at their baseline

exam (July 2000 to August 2002). This study met with the

guidelines of the Declaration of Helsinki. Institutional review

board approval was granted at each study site, and written

informed consent was obtained from all participants. Only

participants with complete retinal, exposure, and covariate

information were included in this analysis.

Retinal Photography and Grading

Retinal photography was performed at each field center using

standardized methods during the second MESA examination,

which took place between August 2002 and January 2004. Of the

6,233 participants that returned for this follow-up exam, 6,176

individuals had retinal photographs taken. For each participant,

the optic disc and macula of both eyes were photographed in a

darkened room using a 45u 6.3-megapixel digital nonmydriatic

camera (Canon), and methods described in detail by Klein and

colleagues [25]. Trained graders who were masked to all

participant characteristics reviewed all images at the University

of Wisconsin Ocular Epidemiology Reading Center in Madison

using previously reported protocols [26–28]. Briefly, retinal vessel

diameters were measured using a computer-based program and

summarized as central retinal arteriolar and venular equivalents

(CRAE and CRVE, respectively). These equivalents represent a

summary of vessel diameters within an area equal to 0.5–1 disc

diameters from the optic disc margin. Vessel diameters for the

right eye were selected for all analyses. Images from the left eye

were used only if retinal vessel diameters could not be graded in

the right eye. The reproducibility of the vessel diameter

measurements in MESA were good, with intragrader and

intergrader correlation coefficients of 0.78 to 0.99 [26].

Participant Characteristics

Detailed data regarding participant health were collected during

the first and second MESA clinical exams, including anthropometry as well as serum levels of high-density lipoprotein (HDL) and

low-density lipoprotein (LDL) cholesterol, glucose, homocysteine,

and inflammatory markers [24]. Information regarding participant

demographics, medical history, and medication use was also

obtained at these exams through technician-administered questionnaires. Residential addresses provided at the exams were

assigned geographic coordinates using ArcGIS software, version

9.1 (ESRI), on the basis of the Dynamap/2000 street network

(Tele Atlas).

City-wide covariates were also collected for each metropolitan

statistical area from the US Census 2000 (http://www.census.gov/

main/www/cen2000.html) and used in sensitivity analyses.

Variables included information about the general population’s

age, race/ethnicity, educational attainment, nativity, unemployment, income, and poverty status. Consistent with the reanalysis of

the Harvard Six Cities and the American Cancer Society Studies

[29], we also explored confounding by city-average elevation,

maximum temperature, and the variation in temperature as

expressed by the standard deviation of daily readings.

Exposure Assignment

Estimates of long-term ambient PM2.5 concentrations were

computed for each participant using a hierarchical spatiotemporal model fit with a pragmatic estimation procedure

described elsewhere [30,31]. The data utilized to derive these

predictions were 2-wk average concentrations of PM2.5 collected

over the course of several years by regulatory monitoring stations

from the US Environmental Protection Agency’s Air Quality

System (AQS) and supplemental monitoring stations specific to

this project [32]. Briefly, the hierarchical model decomposed the

space-time field of concentrations into three pieces: (i) spatially

varying long-term averages, (ii) spatially varying seasonal and longterm trends, and (iii) spatially correlated but temporally independent residuals. Model estimation and prediction were carried out

using a multistep procedure that incorporated a large suite of

spatial covariates such as proximity to major roadways and local

land use to predict average PM2.5 concentrations at each subject’s

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home location over the 2 y preceding the participant’s exam date.

Model selection and validation were carried out using crossvalidation with city-specific cross-validated root mean square

errors for predicting long-term average concentrations at MultiEthnic Study of Atherosclerosis and and Air Pollution (MESA Air)

monitoring locations between 0.34 and 0.94 mg/m3 [30].

Since our hierarchical model is currently only resolved to the 2-

wk time scale, we did not apply this approach for our short-term

analysis. Rather, city-wide average concentrations from AQS

monitoring stations with complete time series during the period of

interest were used to estimate daily concentrations of PM2.5. This

approach is consistent with the majority of acute air pollution

epidemiology research and is justified by the fact that daily PM2.5

concentrations are typically highly correlated across different

locations within a region over time. On the basis of our hypothesis

that acute impacts on the microvasculature would be rapid, we

assigned concentrations for each participant for the day of their

exam, the preceding day, and the average of the preceding 3 d. A

priori, we selected concentrations on the day preceding the exam

for our primary analyses, although we evaluated the other two

time periods in sensitivity analyses.

In other sensitivity analyses, we explored the relationship

between both outcomes and long-term average concentrations of

PM2.5 measured at the nearest AQS monitor as a more traditional

indicator of pollution. Additionally, we evaluated the impact of

living near a major roadway as defined by living within 100 m of

an interstate or US highway (Census Feature Class Code A1 or

A2) or within 50 m of a state or county highway (CFCC A3).

These distances were calculated in ArcGIS 9.1 on the basis of the

TeleAtlas Dynamap 2000 road network.

Data Analysis

Linear regression modeling was performed in R version 2.10.1

(The R Project) in order to examine cross-sectional associations

between air pollution and retinal vessel diameters. Long-and shortterm associations with air pollution concentrations were modeled

independently and jointly. For both CRAE and CRVE, we

performed our modeling in a phased approach and explored

confounding by age, sex, race/ethnicity (white, African American,

Chinese American, Mexican Hispanic, Dominican Hispanic,

Puerto Rican Hispanic, and other Hispanic), and education

(,high school, high school or equivalent, associate, bachelor, or

technical degree, and graduate degree). We also considered

lifestyle factors including smoking status (never, former, and

current), alcohol consumption (user, nonuser), and physical

activity in metabolic equivalent (MET)-minutes/week. In addition,

confounding by BMI, waist to hip ratio, family history

of cardiovascular disease (defined by a history of heart attack,

stroke, or noninjury amputation among a parent, sibling, or child),

history of diabetes mellitus (defined by the 2003 American

Diabetes Association fasting criteria algorithm [33]), systolic

and diastolic blood pressure, emphysema, as well as serum

glucose, HDL and LDL cholesterol, homocysteine, and inflammatory markers (C-reactive protein and fibrinogen) levels was

explored. On the basis of past research, which indicates that

anatomical variation and measurement error in retinal caliber can

lead to spurious associations [34–36], we also explored confounding between CRAE and CRVE. By controlling for the fellow vessel

type in our models, we are further able to address the scientific

question of how air pollution is associated with vessel caliber

relative to the fellow vessel. Although examined, income

(,US$20,000, US$20,000 to ,US$50,000, US$50,000 to

,US$75,000, $US$75,000), secondhand smoke exposures, and

medication use (antihypertensives, lipid-lowering medications,

aspirin, cox-2 inhibitors, erectile dysfunction medications, beta

agonists, sympathomimetics, verapamil, and vasodilators) were

ultimately not included in our main models because there were

more missing data for these parameters and sensitivity analyses

demonstrated little impact on our results. While not of primary

interest, we reported effect estimates for blood pressure and age on

vessel diameters from the fully adjusted models for comparison

purposes.

Linearity of all continuous variables was checked using LOESS

smoothing fits. For CRAE, all variables were modeled as linear

except for systolic blood pressure, which was modeled as a

piecewise linear spline with a knot at 135 mmHg and BMI, which

was modeled as a quadratic. For CRVE, only HDL cholesterol

was modeled with a nonlinear function expressed as a piecewise

linear spline with a knot at 100 mg/dl. For both outcomes,

homocysteine levels were modeled using a log-transformation to

reduce the impact of highly skewed values on the results. For our

acute effect analyses, we included categorical variables for city and

city-specific day of week effects as well as spline fits for city-specific

trends in calendar day (12 degrees of freedom [df]/year),

temperature (6 df), and dew point (6 df) using B-splines to control

for confounding by seasonality and meteorology on the day of the

clinical exam.

To explore the importance of site on our models, we first tested

for statistically significant interactions by city. We then decomposed our PM2.5 concentrations into between- and within-city

exposure contrasts to examine the contribution of each to any

observed associations and investigated results stratified by city.

Finally, we explored the possibility of confounding of our overall

associations by individual city-level factors. We further investigated differential susceptibility by including interaction terms for age,

race/ethnicity, gender, obesity, diabetes, hypertension, use of

specific classes of hypertensive medications, and use of antiinflammatory medications.

Results

With retinal data missing for 283 participants, address or

exposure data missing for 1,104 persons, and covariate data

missing for 243 persons, 4,607 individuals were examined in this

analysis. Descriptive statistics are presented in Table 1 for

individuals with complete information as well as those individuals

excluded from analysis. Included participants were 53% women

among four race/ethnicities (40% white, 26% African American,

12% Chinese American, and 22% Hispanic). Of the Hispanics in

the population, 52%, 14%, 14%, and 35% were of Mexican,

Dominican, Puerto Rican, and other descent, respectively. With a

mean age of 63 y, 44% of the population had hypertension, 13%

had diabetes (an additional 15% had impaired glucose), and 58%

had a family history of cardiovascular disease. Excluded individuals were qualitatively similar to the main cohort although they

were slightly older, of lower socioeconomic position, and had

higher prevalence of hypertension and diabetes.

The mean CRAE in the population was 144 mm and the mean

CRVE was 214 mm (Table 2). Long-term concentrations of outdoor

PM2.5 were similar between our spatio-temporal model predictions

and the closest EPA-monitoring station with means of 16 mg/m3

across all participants and standard deviations of 3 mg/m3. As

anticipated, short-term concentrations were more variable than

long-term averages (standard deviations [SDs] of 9 mg/m3 for the

shortest time frames) and low correlations were found between our

short- and long-term average concentrations (0.25 in crude analyses

and 0 after adjustment for site). Approximately 30% of the

population was living near a major roadway.

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Retinal Arteriolar Diameters

In multivariate regression models, CRAE was negatively

associated with long-term average modeled PM2.5 levels with a

greater magnitude of association seen with increasing control for

potentially confounding variables (Table 3). The strongest effect

estimate was found in the model fully adjusted for all personal,

health, lifestyle characteristics, and CRVE. Similar results were

found in a model that included concentrations of PM2.5 on the day

preceding the health exam. In this final joint model, an interquartile

increase of 3 mg/m3 in our modeled long-term PM2.5 concentrations was associated with a 20.8 mm (95% confidence interval [CI]

21.1 to 20.5) decrease in CRAE. This association was quantitatively similar to a 7-y increase in age or a 3-mm Hg increase in

diastolic blood pressure in our predictive models for CRAE.

Table 1. Demographic and health characteristics of the MESA participants in exam 2.

Characteristics All Participants Participants with Complete Data Excluded Participants

n

Mean (SD) or

Percent n

Mean (SD) or

Percent n

Mean (SD) or

Percent

Personal Characteristics

Age (y) 6,233 64 (10) 4,607 63 (10)a 1,626 66 (11)a

Female (%) 3,241 52 2,439 53 801 49

Race/ethnicity (%)

White 2,493 40 1,853 40 611 38

African American 1,683 27 1,212 26 479 29

Chinese 748 12 537 12 191 12

Hispanic 1,371 22 1,005 22 345 21

Annual income (thousands US$) a a

,20 1,377 23 941 21 451 29

20 to ,50 2,276 38 1,673 38 580 38

50 to ,75 958 16 733 16 214 14

.75 1,377 23 1,107 25 290 19

Education (%) a

Less than high school 994 16 714 15 330 21

High school 2,175 35 1,573 34 564 35

Higher education 1,865 30 1,429 31 445 28

Advanced degree 1,181 19 891 19 269 17

Smoking status (%)

Never 2,847 46 2,149 47 708 45

Former 2,661 43 1,960 43 677 43

Current 681 11 498 11 197 12

Current alcohol user (%) 3,172 51 2,405 52 774 48a

Exercise (MET min/wk) 6,223 1,395 (2,106) 4,607 1450 (2200) 1,615 1,230 (1,800)a

General health characteristics

Waist-hip ratio (%) 6,230 93 (8) 4,607 93 (8) 1,623 94 (8)a

Systolic BP (mm Hg) 6,230 124 (21) 4,607 123 (20)a 1,623 128 (22)a

Diastolic BP (mm Hg) 6,230 70 (10) 4,607 70 (10) 1,623 71 (10)

HDL (mg/dl) 6,183 52 (15) 4,607 52 (15) 1,576 51 (15)

LDL (mg/dl) 6,114 114 (32) 4,607 113 (32) 1,507 114 (33)

Fibrinogen (mg/dl) 6,194 345 (73) 4,607 344 (73) 1,587 350 (72)a

CRP (mgl) 6,191 3.7 (5.4) 4,607 3.6 (5.3) 1,584 3.8 (5.8)

Glucose (mg/dl) 6,184 100 (30) 4,607 99 (28)a 1,577 104 (35)a

Homocysteine (mmol/l) 6,217 9.3 (3.8) 4,607 9.1 (3.5)a 1,610 9.7 (4.1)a

Family history of CVD (%) 3,625 58 2,681 58 944 58

Hypertension (%) 2,838 46 2,008 44a 820 51a

Diabetes (%) 928 15 622 13a 294 19a

Antihypertensive medication (%) 2,508 42 1,808 41 703 45a

Lipid lowering medication (%) 1,313 22 987 22 354 23

a

Statistically significant differences from the main population at the 95% confidence level.

BP, blood pressure; CRP, C-reactive protein; CVD, cardiovascular disease; MET, metabolic equivalent.

doi:10.1371/journal.pmed.1000372.t001

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Figure 1 illustrates the chronic relationship between CRAE and

the underlying trimodal distribution of long-term PM2.5 concentrations in the cohort with clusters of individuals living in St. Paul

and Winston Salem (the lowest polluted cities), New York, Chicago,

and Baltimore (the three modestly polluted cities), and Los Angeles

(the most polluted city). Given the larger variation in long-term

average concentrations between cities (SD = 3.3 mg/m3

) as compared to within-cities (SD = 1.1 mg/m3

), our overall results were

driven by differences in air pollution levels between cities. This

finding is illustrated by Figure 2, which shows the largest city-wide

average retinal diameters among participants in the two least

polluted cities and the smallest city-wide average diameters among

persons in the most polluted city, after control for all confounders in

a model including short-term concentrations. Partitioning the PM2.5

variation into city-wide means and deviations from that mean, we

found that between-city differences in concentration were associated

with a 20.9 mm (95% CI 21.2 to 20.5) decrease in CRAE per

3 mg/m3

. Controlling for site using a random intercepts and

random slopes model, we found that a 3 mg/m3 increase in PM2.5

was associated with a 20.7 mm (95% CI 21.2 to 20.1) decrease in

CRAE. Controlling for site as a fixed effect resulted in a decrease of

20.4 mm (95% CI 21.3 to 0.5) per 3 mg/m3

.

In spite of weaker and less precise estimates within city, we

found no evidence of statistically significant differences in

association by city (p-value for interaction = 0.6). In addition, our

overall results (which include between and within-city variability in

PM2.5) were robust to exclusion of any of the six cities, with the

weakest association found when excluding Baltimore, New York,

or Winston-Salem (20.8 mm [95% CI 21.1 to 20.4] decrease per

3 mg/m3

) and the strongest association found when excluding St.

Paul (21.1 mm [95% CI 21.6 to 20.7] decrease per 3 mg/m3

).

Furthermore, control for a variety of city-wide covariates including

age, race/ethnicity, socioeconomic status, weather, and elevation

did not qualitatively change our findings (unpublished data).We

also found consistency between our modeled estimates of longterm average PM2.5 concentrations and the long-term average

PM2.5 concentrations directly measured at the EPA monitoring

station nearest the participants’ residences, which also had

negative, although generally smaller, associations with CRAE.

Negative associations with larger CIs were also found for

residential proximity to major roadways. In the fully adjusted,

joint models, a 3-mg/m3 increase in PM2.5 measured at the nearest

monitor was associated with a 20.5 mm decrease (95% CI 20.8 to

20.1) in CRAE and living close to a major roadway was

Table 2. Distribution of retinal outcomes and long-term (previous 2 y) and short-term (previous days) exposure estimates among

4,607 participants with complete data.

Variable Mean SD Percentile

Minimum 25th 50th 75th Maximum

Outcome measures

CRAE (mm) 144 14 83 135 144 153 219

CRVE (mm) 214 22 124 199 214 228 324

Long-term exposure estimates

Personal PM2.5 predictions (mg/m3

) 16.3 3.4 9.6 14.6 15.9 17.2 26.3

Nearest monitor PM2.5 (mg/m3

) 16.1 3.3 9.6 12.8 15.9 17.3 25.4

Short-term exposure estimates

Concurrent day PM2.5 (mg/m3

) 15.8 9.3 2.1 9.1 13.7 19.7 69.3

Previous day PM2.5 (mg/m3

) 15.4 9.1 1.4 9.0 13.5 19.6 97.6

Previous 3-d PM2.5 (mg/m3

) 15.2 7.4 3.6 10.0 13.4 18.3 58.6

doi:10.1371/journal.pmed.1000372.t002

Table 3. Associations between retinal diameters and long- (previous 2 y) and short-term (previous day) exposures to fine

particulate air pollution.

Model Retinal Arteriolar Diameter (CRAE) Retinal Venular Diameter (CRVE)

Long-Term Effects Short-Term Effects Long-Term Effects Short-Term Effects

1 20.5 (20.9 to 20.1) 20.4 (20.8 to 20.04) 0.3 (20.3 to 0.9) 20.1 (20.7 to 0.5)

2 20.5 (20.9 to 20.1) 20.6 (21.0 to 20.2) 0.5 (20.04 to 1.1) 20.1 (20.7 to 0.5)

3 20.8 (21.1 to 20.4) 20.5 (21.0 to 0.03) 0.3 (20.2 to 0.9) 20.1 (20.9 to 0.7)

4 20.9 (21.2 to 20.6) 20.4 (20.8 to 0.1) 0.9 (0.4 to –1.4) 0.4 (20.3 to 1.1)

Joint 20.8 (21.1 to 20.5) 20.4 (20.8 to 0.1) 0.9 (0.4 to –1.4) 0.4 (20.3 to 1.1)

All associations reported as mm per interquartile range of 3 mg/m3 (for long term) and 9 mg/m3 (for short term). For both long- and short-term associations, model 1

controlled for age, sex, and race/ethnicity. In our long-term analyses, model 2 also included control for BMI, waist-to-hip ratio, income, education, smoking history,

alcohol use, and family history of cardiovascular disease. Model 3 of our long-term analysis and Model 2 of our short-term analysis added control for LDL, HDL, blood

pressure, diabetes, glucose, physical activity, emphysema, CRP, fibrinogen, and homocysteine. In our short-term analyses, model 2 controlled for all variables in model 3

of our long-term analysis while model 3 also included city-specific trends for day of week, time, temperature, and relative humidity. Model 4 added control for the fellow

vessel diameter (e.g., CRAE or CRVE) to each models 3, and the joint model included long- and short-term concentrations simultaneously with control for covariates

listed in the two respective models 4.

doi:10.1371/journal.pmed.1000372.t003

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associated with a 20.7 mm decrease (95% CI 21.4 to 0.1) in

CRAE.

For short-term concentrations of PM2.5, we found that with

CRAE, a 9-mg/m3 increase in exposure the day prior to the exam

(the interquartile difference) was associated with a 20.4-mm

decrease (95% CI 20.8 to 0.1, p= 0.09) in CRAE in the fully

adjusted model, which included chronic air pollution exposures

(Table 3). These associations were insensitive to adjustment for

long-term concentrations and city, although they were slightly

weakened by control for meteorology and other vessel diameter.

Some sensitivity to exposure window was also found with the

strongest point estimates of decreased diameter generally observed

for our a priori hypothesized exposure period of the previous day.

Decreases of 20.2 mm (95% CI 20.6 to 0.3) and 20.1 mm (95%

CI 20.5 to 0.3) were seen for a 9 mg/m3 increase in the current

day and 7 mg/m3 increase in the previous 3 d concentrations,

respectively.

Only gender appeared to significantly modify the association

between long-term exposures to air pollution and CRAE, with

men demonstrating a larger reduction in retinal diameter

(21.3 mm [95% CI 21.7 to 20.8]) than women (20.4 mm

[95% CI 20.8 to 0.1]) for the same difference (3 mg/m3

) in PM2.5

concentrations. These findings were not found in the short-term

analysis, and no other factors were found to significantly modify

the associations between air pollution and CRAE, including the

presence of diabetes and race/ethnicity.

Retinal Venular Diameters

As hypothesized, long-term PM2.5 concentrations were associated with wider venular diameters (larger CRVE) in our

multivariate models (Table 3). The strongest association was

found in the fully adjusted model, including control for CRAE,

with similar results for the model that included concentrations of

PM2.5 on the day preceding the health exam. An interquartile

increase of approximately 3 mg/m3 in long-term average PM2.5

was associated with a 0.9 mm (95% CI 0.4–1.4 mm) increase in

CRVE. Partitioning the PM2.5 variability into a city mean and

deviations from that mean, we found that between-city differences

were more strongly and significantly associated with CRVE

(1.0 mm [95% CI 0.4–1.5] per 3 mg/m3

) than were pooled withincity differences (0.5 mm [95% CI 20.8 to 1.9] per 3 mg/m3

). In

fact, sensitivity analyses demonstrated that these results were

driven by between city differences on the basis of the cities with the

highest (Los Angeles) and lowest (St. Paul) concentrations

(Figure 3). Exclusion of either of these cities resulted in a 20%–

30% reduction in the effect estimate and a loss of statistical

significance. Like for CRAE, analyses of individual cities

demonstrated highly variable results although St. Paul was found

to have a statistically significant association with CRVE with a

7.0 mm (95% CI 1.5–12.6) increase predicted per 3 mg/m3 in

PM2.5 (results not shown).

Associations derived using modeled concentrations and concentrations measured directly at the nearest EPA monitor were

similar to our spatio-temporal estimates with a 1.1 mm (95% CI

0.6–1.6 mm) increase per 3 mg/m3

. Living near a major roadway

was associated with decreased rather than increased venular

diameter, but this association was not statistically significant in the

fully adjusted model (results not shown). Short-term exposures

were not associated with venular diameters (Table 3).

Discussion

In a large population-based cohort study of adults without

preexisting cardiovascular disease, we found independent associations between long- and short-term concentrations of fine

particulate air pollution and vessel diameters of the retinal

microvasculature, as measured using standardized photographic

methods. Increased air pollution concentrations were associated

with retinal arteriolar narrowing, an outcome that has previously

Figure 1. Associations between retinal arteriolar diameter

(CRAE) and modeled long-term PM2.5 concentrations after

control for covariates. Note: CRAE values represent residuals from

full joint model (i.e., model controlled for age, sex, race/ethnicity, BMI,

waist-to-hip ratio, income, education, smoking history, alcohol use,

family history of cardiovascular disease, LDL, HDL, blood pressure,

diabetes, glucose, physical activity, emphysema, CRP, fibrinogen,

homocysteine, CRVE, and previous day PM2.5 concentration). Data are

plotted as a cubic polynomial with 3 df.

doi:10.1371/journal.pmed.1000372.g001

Figure 2. City-wide associations between CRAE and modeled

long-term PM2.5 concentrations after control for covariates.

Notes: CRAE values presented as the residuals from a model controlled

for age, sex, race/ethnicity, BMI, waist-to-hip ratio, income, education,

smoking history, alcohol use, family history of cardiovascular disease,

LDL, HDL, blood pressure, diabetes, glucose, physical activity, emphysema, CRP, fibrinogen, homocysteine, CRVE, and previous day PM2.5

concentration.

doi:10.1371/journal.pmed.1000372.g002

Retinal Microvasculature and Pollution

PLoS Medicine | www.plosmedicine.org 7 November 2010 | Volume 7 | Issue 11 | e1000372
been associated with increased risk of myocardial infarction,

stroke, hypertension, and cardiovascular mortality, independent of

other traditional risk factors. [13–19].

An increase of 3 mg/m3 in long-term PM2.5 concentrations

predicted at participant homes was associated with a 20.8 mm or

20.6% decrease in arteriolar diameter. This decrease is of the

same order of magnitude as that of more traditional risk factors,

with reductions equivalent to those seen for a 7-y increase in age or

a 3-mm Hg increase in diastolic blood pressure in this cohort.

Daily increases in PM2.5 concentrations were similarly associated

with narrower arteriolar diameters, though these associations were

not statistically significant (p= 0.09). All associations persisted after

control for hypertension and other factors previously reported to

be associated with narrower retinal arterioles and venular

diameters. Although positive associations were demonstrated

between chronic exposures to ambient PM2.5 and venular

diameters, increases that have been associated with risk of stroke

[13,19], these relationships were less robust and less consistent

across exposure metrics than those found for CRAE.

This investigation is, to our knowledge, the first study to directly

examine the relationship of ambient air pollution and in vivo

measures of the human microvasculature. This study adds to

previous in vivo work on the impacts of air pollution focused on

larger vessels and extends past investigations on the ocular

manifestations of environmental exposures [37–40] by evaluating

them in a population-based cohort with high quality information

on exposure, outcome, and confounders. Our findings support the

hypothesis that subclinical microvascular changes are associated

with PM exposures, even within the range of concentrations

common in many developed countries with adopted pollution

control measures and at levels much lower than are often found in

newly industrialized urban centers. Finally, jointly modeling the

short- and long-term impacts of a pollutant on vascular health

provides new evidence that both time frames may be relevant for

this outcome and enlarges the limited knowledge base in an area of

substantial scientific uncertainty.

Our results are generally consistent with the existing literature

on air pollution and the human vasculature, which has thus far

examined associations with vessel diameter and function in the

forearm. Several studies have used ultrasound to study the brachial

artery, a conduit vessel with distinct anatomical and functional

properties. In experimental settings, short-term exposures to high

levels of air pollutants have been shown to elicit vasoconstriction of

the brachial artery but not impair endothelium-dependent dilation

[12,41–43]. In observational studies, short-term exposures have

been associated with impaired endothelium-dependent vasodilation [9,11]. Though the brachial artery assesses endothelial

function in a vessel comparable to the epicardial coronary arteries,

it has not been shown, to date, to be generalizable to the

microvasculature of the heart and lungs, which are critically

important in the progression of clinical vascular disease [44].

Other studies, which have examined an amalgamation of vessels

smaller than the brachial artery using forearm plethysmography

have similarly shown diminution in forearm blood flow following

short-term exposure to diesel exhaust [10,45]. Furthermore, recent

toxicological investigations have demonstrated that acute exposures to PM blunt arteriolar dilatation of skeletal and subepicardial

muscle in rats. These studies also demonstrated increased

leukocyte adhesion and rolling in paired venules, elevated

leukocyte deposition, increased oxidative stress, and decreased

bioavailability of nitric oxide [7,44,46,47]. Research also has

suggested that air pollution can contribute to elevated blood

pressure in humans [43,48].

Our findings of widened venular diameters with chronic

exposures to air pollution are similarly consistent with the

literature based on investigations of cigarette smoke [23,49].

Although the biological mechanisms for these associations remain

to be fully elucidated, it is thought that CRVE widening might be,

at least partially, due to systemic inflammation [23]. Nevertheless,

our observed associations for CRVE should be interpreted with

caution as they were not robust to exposure parameterization or

timing of exposure, were driven entirely by differences between the

two cities with the highest and lowest ambient air pollution

concentrations, and were sensitive to control for CRAE.

Taken together, our findings support an increasingly consistent

view of the downstream mechanisms through which particulate

matter exposures might cause the clinical cardiovascular disease

observed to be associated with air pollution in epidemiological

studies. As such, these results further enhance the biological

plausibility of observed air pollution–disease relationships. Although we cannot directly identify the upstream pathways of short

and long-term exposures to PM2.5 that might have caused the

observed narrowing of retinal arterioles, the fact that our findings

persisted following adjustment for blood pressure and hypertension status may imply that these microvasculature changes are not

a response to other changes in vascular resistance.

Our findings of significantly narrower retinal arteriolar

diameters (and significant widening of retinal venular diameters)

among persons residing in areas with higher long-term concentrations of air pollution are unique and support many published

findings of elevated cardiovascular mortality risk in areas of

elevated pollution. [50–52]. The observation of independent

associations with microvascular phenomenon for short-term and

long-term concentrations of PM2.5 is consistent with an impact of

chronic pollution exposure that exceeds what can be ascribed to

short-term increases in pollutant concentrations. Although we

cannot fully exclude the possibility of confounding between shortand long-term associations without temporally resolved spatial

predictions on a shorter time scale, this joint modeling method

may help to further disentangle acute and long-term relationships

with the cardiovascular system.

It should be noted that our long-term exposure findings were

driven by between-city differences in pollution concentrations,

Figure 3. City-wide associations between CRVE and modeled

long-term PM2.5 concentrations after control for covariates.

Notes: CRVE values presented as the residuals from a model controlled

for age, sex, race/ethnicity, BMI, waist-to-hip ratio, income, education,

smoking history, alcohol use, family history of cardiovascular disease,

LDL, HDL, blood pressure, diabetes, glucose, physical activity, emphysema, CRP, fibrinogen, homocysteine, CRAE, and previous day PM2.5

concentration.

doi:10.1371/journal.pmed.1000372.g003

Retinal Microvasculature and Pollution

PLoS Medicine | www.plosmedicine.org 8 November 2010 | Volume 7 | Issue 11 | e1000372
most likely as the result of enhanced variability in PM2.5

concentrations between cities as compared to within any city.

Our findings, however, are supported by a nonsignificant negative

association with our pooled estimate of within-city variability, a

lack of sensitivity of the arteriolar results to exclusion of any one

particular city, and consistent negative associations for short-term

fluctuations in pollution that are independent of city. The

observed associations are also unlikely to be due to differences in

data collection as standardized methods were used to maintain

consistency across sites in retinal image collection. Similarly, all

measures were read in a masked manner by a single centralized

reading center using a validated protocol to standardize the

grading of image parameters. Finally, our results also were found

to be qualitatively similar across models with differing levels

of control for many potential confounders (individual-level and

city-wide).

Another point to note is that there was a relatively high

proportion of missing data (16%) as a result of incomplete

participant geocoding. Although individuals in rural or newer

areas might be less likely to be geocoded, we do not anticipate any

bias to our analysis since the likelihood of data being missing

should be unrelated to the outcome.

Overall, these findings may have practical relevance since

relationships with subclinical microvascular changes were found at

PM2.5 levels that commonly occur in developed nations and well

below those in developing countries. With a mean long-term

concentration of 16 mg/m3

, these levels were similar to the annual

National Ambient Air Quality Standard of 15 mg/m3 established

by the US EPA for PM2.5. The observation of a linear trend in the

relationship between PM2.5 and CRAE further supports past

findings [6], which have failed to find evidence of a threshold and

suggest that the cardiovascular effects of air pollution can occur at

levels below existing regulatory standards. Since different airborne

pollutants can be correlated in the environment, we cannot

exclude the possibility that these results might also reflect the

toxicity of another correlated pollutant or the pollution mixture as

a whole. As the MESA Air project proceeds with additional

refinements to the air pollution estimates, including indoor

predictions and investigation of other copollutants, and the MESA

study collects an additional round of retinal photography in this

cohort, we will be able to confirm our findings and examine this

phenomenon in greater detail.

Conclusion

Long- and short-term concentrations of fine particulate matter

were associated with observable differences in microvascular

structure in a cross-sectional analysis of a large cohort,

characterized by high quality information on exposure, outcome,

and potential confounders. Individuals with higher long- and

short-term air pollution concentrations had narrower arteriolar

diameters, even after control for typical risk factors. We also found

associations between chronic but not acute air pollution levels and

widened retinal venules, another marker of microvascular disease,

although these associations were less robust. Overall, our findings

enhance the biological plausibility of the hypothesis that important

vascular phenomena are associated with small increases in shortterm or long-term air pollution exposures, even at current

exposure levels. These results further support reported associations

between air pollution and the development and exacerbation of

clinical cardiovascular disease.

Acknowledgements

The authors are grateful for the other investigators, staff, and participants

of the MESA, MESA Eye, and MESA Air studies for their valuable

contributions to this work. A full list of MESA investigators and institutions

can be found at http://www.mesa-nhlbi.org.

Author Contributions

ICMJE criteria for authorship read and met: SDA RK BEKK AAS MFC

TYW MSO SS RGB DSS MLD PDS JDK. Agree with the manuscript’s

results and conclusions: SDA RK BEKK AAS MFC TYW MSO SS RGB

DSS MLD PDS JDK. Designed the experiments/the study: SDA MFC

DSS JDK. Analyzed the data: SDA AAS SS. Collected data/did

experiments for the study: RK BEKK SS RGB MLD. Enrolled patients:

RGB MLD. Wrote the first draft of the paper: SDA. Contributed to the

writing of the paper: SDA RK BEKK AAS MFC TYW MSO SS RGB

DSS MLD PDS JDK. Critically revised manuscript for important

intellectual content: BEKK. Developed statistical models: AAS. Helped

to develop the collaboration with MESA that led to MESA AIR, and

helped obtain funding for MESA AIR from the EPA: DSS. Reviewed the

results of various analyses and suggested additional analyses: DSS.

Responsible for the spatio-temporal model and estimates of exposure: PDS.

References

1. Brook RD, Rajagopalan S, Pope CA, Brook JR, Bhatnagar A, et al. (2010)

Particulate matter air pollution and cardiovascular disease: an update to the

scientific statement from the American Heart Association. Circulation 121:

2331–2378.

2. Hoek G, Brunekreef B, Goldbohm S, Fischer P, van den Brandt PA (2002)

Association between mortality and indicators of traffic-related air pollution in the

Netherlands: a cohort study. Lancet 360: 1203–1209.

3. Maheswaran R, Elliott P (2003) Stroke mortality associated with living

near main roads in England and Wales - A geographical study. Stroke 34:

2776–2780.

4. Tonne C, Melly S, Mittleman M, Coull B, Goldberg R, et al. (2007) A casecontrol analysis of exposure to traffic and acute myocardial infarction. Environ

Health Perspect 115: 53–57.

5. Adar SD, Kaufman JD (2007) Cardiovascular disease and air pollutants:

evaluating and improving epidemiological data implicating traffic exposure.

Inhal Toxicol 19: 135–149.

6. Pope CA, Dockery DW (2006) Health effects of fine particulate air pollution:

lines that connect. Air Waste 56: 709–742.

7. Nurkiewicz TR, Porter DW, Barger M, Castranova V, Boegehold MA (2004)

Particulate matter exposure impairs systemic microvascular endotheliumdependent dilation. Environ Health Perspect 112: 1299–1306.

8. Mills NL, Tornqvist H, Gonzalez MC, Vink E, Robinson SD, et al. (2007)

Ischemic and thrombotic effects of dilute diesel-exhaust inhalation in men with

coronary heart disease. N Engl J Med 357: 1075–1082.

9. Rundell KW, Hoffman JR, Caviston R, Bulbulian R, Hollenbach AM (2007)

Inhalation of ultrafine and fine particulate matter disrupts systemic vascular

function. Inhal Toxicol 19: 133–140.

10. Mills NL, Tornqvist H, Robinson SD, Gonzalez M, Darnley K, et al. (2005)

Diesel exhaust inhalation causes vascular dysfunction and impaired endogenous

fibrinolysis. Circulation 112: 3930–3936.

11. O’Neill MS, Veves A, Zanobetti A, Sarnat JA, Gold DR, et al. (2005) Diabetes

enhances vulnerability to particulate air pollution - Associated impairment in

vascular reactivity and endothelial function. Circulation 111: 2913–2920.

12. Brook RD, Brook JR, Urch B, Vincent R, Rajagopalan S, et al. (2002)

Inhalation of fine particulate air pollution and ozone causes acute arterial

vasoconstriction in healthy adults. Circulation 105: 1534–1536.

13. Wong TY, Kamineni A, Klein R, Sharrett AR, Klein BE, et al. (2006)

Quantitative retinal venular caliber and risk of cardiovascular disease in older

persons - The Cardiovascular Health Study. Arch Intern Med 166: 2388–2394.

14. Witt N, Wong TY, Hughes AD, Chaturvedi N, Klein BE, et al. (2006)

Abnormalities of retinal microvascular structure and risk of mortality from

ischemic heart disease and stroke. Hypertension 47: 975–981.

15. Wong TY, Klein R, Nieto FJ, Klein BEK, Sharrett AR, et al. (2003) Retinal

microvascular abnormalities and 10-year cardiovascular mortality - A population-based case-control study. Ophthalmology 110: 933–940.

16. Duan YK, Mo JP, Klein R, Scott IU, Lin HM, et al. (2007) Age-related macular

degeneration is associated with incident myocardial infarction among elderly

Americans. Ophthalmology 114: 732–737.

Retinal Microvasculature and Pollution

PLoS Medicine | www.plosmedicine.org 9 November 2010 | Volume 7 | Issue 11 | e1000372
17. Wong TY, Klein R, Couper DJ, Cooper LS, Shahar E, et al. (2001) Retinal

microvascular abnormalities and incident stroke: the atherosclerosis risk in

communities study. Lancet 358: 1134–1140.

18. Wong TY, Klein R, Sharrett AR, Duncan BB, Couper DJ, et al. (2002) Retinal

arteriolar narrowing and risk of coronary heart disease in men and women - The

atherosclerosis risk in communities study. JAMA 287: 1153–1159.

19. McGeechan K, Liew G, Macaskill P, Irwig L, Klein R, et al. (2009) Prediction of

incident stroke events based on retinal vessel caliber: a systematic review and

individual-participant meta-analysis. Am J Epidemiol 170: 1323–1332.

20. Cheung N, Bluemke DA, Klein R, Sharrett AR, Islam FMA, et al. (2007)

Retinal arteriolar narrowing and left ventricular remodeling - The multi-ethnic

study of atherosclerosis. J Am Coll Cardiol 50: 48–55.

21. Cheung N, Islam FMA, Jacobs DR, Sharrett AR, Klein R, et al. (2007) Arterial

compliance and retinal vascular caliber in cerebrovascular disease. Ann Neurol

62: 618–624.

22. Cheung N, Sharrett AR, Klein R, Criqui MH, Islam A, et al. (2007) Aortic

distensibility and retinal arteriolar narrowing - The multi-ethnic study of

atherosclerosis. Hypertension 50: 617–622.

23. Wong TY, Islam FMA, Klein R, Klein BEK, Cotch MF, et al. (2006) Retinal

vascular caliber, cardiovascular risk factors, and inflammation: The Multi-Ethnic

Study of Atherosclerosis (MESA). Invest Ophthalmol Vis Sci 47: 2341–2350.

24. Bild DE, Bluemke DA, Burke GL, Detrano R, Roux AVD, et al. (2002) Multiethnic study of atherosclerosis: objectives and design. Am J Epidemiol 156: 871–881.

25. Klein R, Klein BEK, Knudtson MD, Wong TY, Cotch MF, et al. (2006)

Prevalence of age-related macular degeneration in 4 racial/ethnic groups in the

multi-ethnic study of atherosclerosis. Ophthalmology 113: 373–380.

26. Wong TY, Knudtson MD, Klein R, Klein BEK, Meuer SM, et al. (2004)

Computer-assisted measurement of retinal vessel diameters in the beaver dam

eye study - Methodology, correlation between eyes, and effect of refractive

errors. Ophthalmology 111: 1183–1190.

27. Knudtson MD, Lee KE, Hubbard LD, Wong TY, Klein R, et al. (2003) Revised

formulas for summarizing retinal vessel diameters. Curr Eye Res 27: 143–149.

28. Hubbard LD, Brothers RJ, King WN, Clegg LX, Klein R, et al. (1999) Methods

for evaluation of retinal microvascular abnormalities associated with hypertension/sclerosis in the atherosclerosis risk in communities study. Ophthalmology

106: 2269–2280.

29. Health Effects Institute (2000) Reanalysis of the Harvard Six Cities Study and

the American Cancer Society Study of Particulate Air Pollution and Mortality.

Boston: Health Effects Institute.

30. Sampson PD, Szpiro AA, L. S, JL, Kaufman JD (2009) Pragmatic estimation of

a spatio-temporal air quality model with irregular monitoring data. UW

Biostatistics Working Paper Series Working Paper 353.

31. Szpiro A, Sampson P, Sheppard L, Lumley T, Adar S, et al. (2009) Predicting

intra-urban variations in air pollution with complex spatio-temporal dependencies. Environmetrics 21: 606–631.

32. Cohen MA, Adar SD, Allen RW, Avol E, Curl CL, et al. (2009) Approach to

estimating participant pollutant exposures in the Multi-Ethnic Study of

Atherosclerosis and Air Pollution (MESA Air). Environ Sci Technol 43:

4687–4693.

33. Genuth S, Alberti K, Bennett P, Buse J, DeFronzo R, et al. (2003) Follow-up

report on the diagnosis of diabetes mellitus. Diabetes Care 26: 3160–3167.

34. Liew G, Sharrett AR, Kronmal R, Klein R, Wong TY, et al. (2007)

Measurement of retinal vascular caliber: Issues and alternatives to using the

arteriole to venule ratio. Invest Ophthalmol Vis Sci 48: 52–57.

35. Liew G, Sharrett AR, Wang JJ, Klein R, Klein BEK, et al. (2008) Relative

importance of systemic determinants of retinal arteriolar and venular caliber.

Arch Ophthal 126: 1404–1410.

36. Liew G, Wong TY, Mitchell P, Wang JJ (2006) Are narrower or wider retinal

venules associated with incident hypertension? Hypertension 48: E10–E10.

37. Lois N, Abdelkader E, Reglitz K, Garden C, Ayres JG (2008) Environmental

tobacco smoke exposure and eye disease. Br J Ophthalmol 92: 1304–1310.

38. Memisogullari R, Yuksel H, Coskun A, Yazgan O, Bilgin C (2007) High serum

homocysteine levels correlate with a decrease in the blood flow velocity of the

ophthalmic artery in highway toll collectors. Tohoku J Exp Med 212: 247–252.

39. Remky A, Dressen D, Plange N, Korinth G, Drexler H, et al. (2004) Retinal

vessel diameters determined in an ergo-medical field study: diameter changes

associated with the occupational exposure to carbon disulfide. Association

Research Vision Ophthalmology Inc. 45: E-Abstract 5253.

40. Sliwka U, Krasney JA, Simon SG, Schmidt P, Noth J (1998) Part one: Effects of

sustained low-level elevations of carbon dioxide on cerebral blood flow and

autoregulation of the intracerebral arteries in humans. Aviat Space Environ Med

69: 299–306.

41. Urch B, Brook JR, Wasserstein D, Brook RD, Rajagopalan S, et al. (2004)

Relative contributions of PM2.5 chemical constituents to acute arterial

vasoconstriction in humans. Inhal Toxicol 16: 345–352.

42. Peretz A, Kaufman JD, Trenga CA, Allen J, Carlsten C, et al. (2008) Effects of

diesel exhaust inhalation on heart rate variability in human volunteers. Environ

Res 107: 178–184.

43. Brook RD, Urch B, Dvonch JT, Bard RL, Speck M, et al. (2009) Insights into

the mechanisms and mediators of the effects of air pollution exposure on blood

pressure and vascular function in healthy humans. Hypertension 54: 659–U420.

44. LeBlanc AJ, Cumpston JL, Chen BT, Frazer D, Castranova V, et al. (2009)

Nanoparticle inhalation impairs endothelium-dependent vasodilation in subepicardial arterioles. J Toxicol Environ Health A 72: 1576–1584.

45. To ̈rnqvist H, Mills NL, Gonzales M, Miller MR, Robinson SD, et al. (2007)

Persistent endothelial dysfunction following diesel exhaust inhalation in man.

Am J Respir Crit Care Med 176: 395–400.

46. Nurkiewicz TR, Porter DW, Barger M, Millecchia L, Rao KMK, et al. (2006)

Systemic microvascular dysfunction and inflammation after pulmonary

particulate matter exposure. Environ Health Perspect 114: 412–419.

47. Nurkiewicz TR, Porter DW, Hubbs AF, Stone S, Chen BT, et al. (2009)

Pulmonary nanoparticle exposure disrupts systemic microvascular nitric oxide

signaling. J Toxicol Sci 110: 191–203.

48. Auchincloss AH, Roux AVD, Dvonch JT, Brown PL, Barr RG, et al. (2008)

Associations between recent exposure to ambient fine particulate matter and

blood pressure in the Multi-Ethnic Study of Atherosclerosis (MESA). Environ

Health Perspect 116: 486–491.

49. Kifley A, Liew G, Wang JJ, Kaushik S, Smith W, et al. (2007) Long-term effects

of smoking on retinal microvascular caliber. Am J Epidemiol 166: 1288–1297.

50. Dockery DW, Pope CA, 3rd, Xu X, Spengler JD, Ware JH, et al. (1993) An

association between air pollution and mortality in six U.S. cities. N Engl J Med

329: 1753–1759.

51. Pope CA, Burnett RT, Thurston GD, Thun MJ, Calle EE, et al. (2004)

Cardiovascular mortality and long-term exposure to particulate air pollution -

Epidemiological evidence of general pathophysiological pathways of disease.

Circulation 109: 71–77.

52. Miller KA, Siscovick DS, Sheppard L, Shepherd K, Sullivan JH, et al. (2007)

Long-term exposure to air pollution and incidence of cardiovascular events in

women. N Engl J Med 356: 447–458.

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Editors’ Summary

Background. Cardiovascular disease (CVD)—disease that

affects the heart and/or the blood vessels—is a common

cause of illness and death among adults in developed

countries. In the United States, for example, the leading

cause of death is coronary heart disease, a CVD in which

narrowing of the heart’s arteries by atherosclerotic plaques

(fatty deposits that build up with age) slows the blood

supply to the heart and may eventually cause a heart attack

(myocardial infarction). Other types of CVD include stroke (in

which atherosclerotic plaques interrupt the brain’s blood

supply) and peripheral arterial disease (in which the blood

supply to the limbs is blocked). Smoking, high blood

pressure, high blood levels of cholesterol (a type of fat),

having diabetes, being overweight, and being physically

inactive all increase a person’s risk of developing CVD.

Treatments for CVD include lifestyle changes and taking

drugs that lower blood pressure or blood cholesterol levels.

Why Was This Study Done? Another risk factor for CVD is

exposure to long-term and/or short-term air pollution. Fine

particle pollution or PM2.5 is particularly strongly associated

with an increased risk of CVD. PM2.5—particulate matter

2.5 mm in diameter or 1/30th the diameter of a human hair—

is mainly produced by motor vehicles, power plants, and

other combustion sources. Why PM2.5 increases CVD risk is

not clear but one possibility is that it alters the body’s

microvasculature (fine blood vessels known as capillaries,

arterioles, and venules), thereby impairing the blood flow

through the heart and brain. In this study, the researchers

use noninvasive digital retinal photography to investigate

whether there is an association between air pollution and

changes in the human microvasculature. The retina—a lightsensitive layer at the back of the eye that converts images

into electrical messages and sends them to the brain—has a

dense microvasculature. Retinal photography is used to

check the retinal microvasculature for signs of potentially

blinding eye diseases such as diabetic retinopathy. Previous

studies have found that narrower than normal retinal

arterioles and wider than normal retinal venules are

associated with CVD.

What Did the Researchers Do and Find? The researchers

used digital retinal photography to measure the diameters of

retinal blood vessels in the participants of the Multi-Ethnic

Study of Atherosclerosis (MESA). This study is investigating

CVD progression in people aged 45–84 years of various

ethnic backgrounds who had no CVD symptoms when they

enrolled in the study in 2000–2002. The researchers modeled

the long-term outdoor concentration of PM2.5 at each

participant’s house for the 2-year period preceding the

retinal examination (which was done between 2002 and

2003) using data on PM2.5 levels collected by regulatory

monitoring stations as well as study-specific air samples

collected outside of the homes and in the communities of

study participants. Outdoor PM2.5 measurements taken the

day before the examination provided short-term PM2.5 levels.

Among the 4,607 MESA participants who had complete data,

retinal arteriolar diameters were narrowed among those who

lived in regions with increased long- and short-term PM2.5

levels. Specifically, an increase in long-term PM2.5

concentrations of 3 mg/m3 was associated with a 0.8 mm

decrease in arteriolar diameter, a reduction equivalent to

that seen for a 7-year increase in age in this group of people.

Living near a major road, another indicator of long-term

exposure to PM2.5 pollution, was also associated with

narrowed arterioles. Finally, increased retinal venular

diameters were weakly associated with long-term high

PM2.5 concentrations.

What Do These Findings Mean? These findings indicate

that living in areas with long-term air pollution or being

exposed to short-term air pollution is associated with

narrowing of the retinal arterioles in older individuals. They

also show that widening of retinal venules is associated with

long-term (but not short-term) PM2.5 pollution. Together,

these findings support the hypothesis that long- and shortterm air pollution increases CVD risk through effects on the

microvasculature. However, they do not prove that PM2.5 is

the constituent of air pollution that drives microvascular

changes—these findings could reflect the toxicity of another

pollutant or the pollution mixture as a whole. Importantly,

these findings show that microvascular changes can occur at

the PM2.5 levels that commonly occur in developed

countries, which are well below those seen in developing

countries. Worryingly, they also suggest that the deleterious

cardiovascular effects of air pollution could occur at levels

below existing regulatory standards.

Additional Information. Please access these Web sites via

the online version of this summary at http://dx.doi.org/

10.1371/journal.pmed.1000372.

N The American Heart Association provides information for

patients and caregivers on all aspects of cardiovascular

disease (in several languages), including information on air

pollution, heart disease, and stroke

N The US Centers for Disease Control and Prevention has

information on heart disease and on stroke

N Information is available from the British Heart Foundation

on cardiovascular disease

N The UK National Health Service Choices website provides

information for patients and caregivers about cardiovascular disease

N MedlinePlus provides links to other sources of information

on heart disease and on vascular disease (in English and

Spanish)

N The AIRNow site provides information about US air quality

and about air pollution and health

N The Air Quality Archive has up-to-date information about

air pollution in the UK and information about the health

effects of air pollution

N The US Environmental Protection Agency has information

on PM2.5

N The following Web sites contain information available on

the MESA and MESA Air studies

Retinal Microvasculature and Pollution

PLoS Medicine | www.plosmedicine.org 11 November 2010 | Volume 7 | Issue 11 | e1000372