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Association between Benzene Exposure, Circulating Angiogenic Cell Levels, and Cardiovascular Disease Risk in the Louisville Healthy Heart Study

APHA Annual Meeting, 17 November 2014

Image: http://ehp.niehs.nih.gov/realfiles/docs/1998/106-11/forumfigheart.JPG

Natasha DeJarnett,1 Daniel J. Conklin,1 Daniel Riggs,1 Timothy E. O’Toole,1

James McCracken,1 Wes Abplanalp,1 Petra Haberzettl,1 Ray Yeager,2

Sanjay Srivastava,1 Ihab Hamzeh,1 Stephen Wagner,1 Atul Chugh,1

Andrew DeFilippis,1 Tiffany Ciszewski,1 Bradley Wyatt,1 Carrie Becher,1

Deirdre Higdon,1 Zhengzhi Xie,1 Kenneth Ramos,3 David J. Tollerud,2 John Myers,4

Shesh Rai,5 Sumanth D. Prabhu,1 Aruni Bhatnagar1,3

University of Louisville Division of Cardiovascular Medicine,1 Environmental and Occupational Health Sciences,2 Biochemistry and Molecular Biology,3 Pediatrics,4 and Biostatistics and Bioinformatics5

Overview

• Background

• Cardiovascular disease (CVD) burden

• CVD and pollution

• Endothelial progenitor cells (EPCs)/ Circulating angiogenic cells (CACs)

• Benzene exposure effects

• Methods of the Healthy Heart Study

• Results

• Summary

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Circulating AngiogenicCell Levels

Endothelial Dysfunction

BenzeneCVD

Air Pollution

Burden of Cardiovascular Disease

• Cardiovascular disease (CVD)

• 1 in 3 US adults have CVD1

• By 2030, over 40% of people are predicted to have CVD2

• Lifetime risk of 52% (men) and 39% (women) at age 501

• US expenses exceed $400 billion3

• Air pollution increases CVD risk

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1) Go et al. 20132) Heidenreich et al. 20113) Mensah and Brown, 2007

6

6.2

7.4

9.3

10.8

11.6

14.4

21.4

22.0

25.3

37.3

43.3

53.5

190.1

232.3

Homicide

Parkinson's

Essential hypertension/hypertensive renal…

Chronic liver disease and cirrhosis

Suicide

Septicemia

Nephritis, nephrotic syndrome, nephrosis

Alzheimer's

Influenza and pneumonia

Diabetes

Accidents

Chronic lower respiratory diseases

Stroke

Cancer

Diseases of the Heart

Age-Adjusted Death Rates (per 100,000 population) for the 15 Leading Causes of Death, 2003

Mensah and Brown 2007

36.9

37.8

38.7

39.7

40.5

35

36

37

38

39

40

41

2010 2015 2020 2025 2030

PR

EVA

LEN

CE

YEAR

Projected US CVD Prevalence

Heidenreich et al. 2011Nawrot et al. 2011

7.4%

4.8%

Background Methods Results Discussion

Biological Plausibility

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Background Methods Results Discussion

Endothelial Progenitor Cells (EPCs)

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Shantsila et al. 2007

Marker Differentiation

CD34+ Stem

CD133+ Early Stem

CD45+ Hematopoietic

CD31+ EndothelialJujo et al. 2008

Fadini et al. 2007, 2012

Background Methods Results Discussion

EPCs and CVD Risk

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Vasa et al. 2001 Hill et al. 2003

Effect of risk factors on number of EPCs

Association between the FRS

and EPC Colony Counts

Background Methods Results Discussion

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O’Toole et al. 2010

EPCs/CACs and Pollution

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Background Methods Results Discussion

Association between Human EPCs and PM2.5 Association between Mouse EPCs and PM2.5

Association between Human Circulating Angiogenic Cells (CACs) and Acrolein

O’Toole et al. 2010

DeJarnett et al. 2014

Haberzettl et al. 2012

Benzene

Benzene-CVD Risk• Bone marrow toxicant

• Associated with increased cardiovascular mortality4

• Chronic exposure linked with hypertension5, 6

• Found in high amounts in traffic, industrial emissions, and cigarette smoke

• Benzene metabolite trans, trans-muconic acid (t,t-MA) linked with environmental exposure

Benzene Metabolism

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Background Methods Results Discussion

Cytochrome P450 (CYP)Badham et al. 2010

4) Tsai et al. 20105) Kotseva and Popov 19986) Wiwanitkit 2007

Traffic Proximity

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Background Methods Results Discussion

Hoffmann et al. 2007

Traffic Proximity Increases Odds for Atherosclerosis

Objective

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Background Methods Results Discussion

Experimental Procedures

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Cross-sectional Design

• Patient recruitment from UofL AIM and Preventive Cardiology clinics 10/2009 – 03/2011

• Receiving primary or secondary CVD care

• Questionnaire, blood and urine collection during exam

CAC Identification

• Flow cytometry identified circulating angiogeniccells (CACs) with antigenic markers• CD31+ (endothelial)

• CD34+ (stem)

• CD45+ (hematopoietic)

• AC133+/– (early/late stem)

• Id-1+ verified angiogeniccells

Benzene Exposure

• t,t-MA and tobacco metabolites quantified by GC/MS

• Self-report of tobacco exposure

• Distance to major roadway (10,000 vehicles/day) assessed using Geographic Information Systems (GIS)

Statistical Analysis

• Descriptive stats

• Transform CACs

• T-tests, ANOVA

• Χ2 Analysis

• Generalized linear models

Background Methods Results Discussion

CAC Identification

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CAC Population

Cell Differentiation State Abundance

Cell type-1 CD31+/34+/45dim 0.2%

Cell type-2 CD31+/34+/45+ 0.1%

Cell type-3 CD31+/34+/45dim/AC133+ 0.1%

Cell type-4 CD31+/34+/45+/AC133+ 0.01%

Cell type-5 CD31+/AC133+ 0.3%

Cell type-6 CD31+/34+ 0.4%

Cell type-7 CD31+/34+/45dim/AC133– 0.2%

Cell type-8 CD31+/34+/45+/AC133– 0.1%

Cell type-9 CD34+ 0.3%

Cell type-10 CD31+ 32%

Cell type-11 AC133+ 0.4%

Cell type-12 CD45+ 55%

Cell type-13 CD34+/AC133+ 0.1%

Cell type-14 CD34+/45+/AC133+ 0.01%

Cell type-15 CD34+/45dim/AC133+ 0.1%

Cell type-14

Cell type-8 (–) ControlCD31+/34+/45dim

Cell type-2

Cell type-11

Figure 1. Id-1

stains of Cell

types 2, 8, 11,

and 14. Presence

of Id-1 (green

stain) in nuclei

(blue stain)

indicates validated

angiogenic cells.

Background Methods Results Discussion

Table 1. Antigenic identity of CACs.

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Table 2. Demographic summary of the Louisville Healthy Heart Study stratified by t,t-MA tertiles. Low t,t-MA ≤0.06;

medium t,t-MA >0.06–0.16, high t,t-MA >0.16 mg/g creatinine. Revascularization includes coronary artery bypass graft,

percutaneous coronary intervention, or stents. Platelet-leukocyte aggregates are the % total of cells CD41+/45+. * =

significant at the 0.05 level.

Background Methods Results Discussion

Smoking and Benzene

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β=0.087, R2 = 0.069, p<0.001

Nonsmokers

Smokers

Figure 2A. Positive association between cotinine

and t,t-MA. Cotinine levels were regressed against

t,t-MA, verifying the link between smoking and

benzene exposure.

Background Methods Results Discussion

β=0.315, R2=0.135, p<0.001A B

Nonsmokers

Smokers

Figure 2B. Positive association between t,t-MA

and 3-HPMA. 3-HPMA levels were regressed

against t,t-MA, further verifying the link between

smoking and benzene exposure.

Roadway and Benzene

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Figure 3B. No association between t,t-MA and

distance from a major roadway. Residential

proximity to a major road (annual mean of

10,000 vehicles/day).

β=-0.129, R2=0.013, p=0.134

Figure 3A. Healthy Heart Study geographic

distribution. Participant residences are

geographically masked.

Background Methods Results Discussion

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* p < 0.05 † Nonsmokers have cotinine < 200µg/g creatinine.

Table 3. Significant associations (adjusted) between t,t-MA and circulating

angiogenic cell levels.

t,t-MA RegressionCell type-2

(CD31+/34+/45+)

Cell type-8

(CD31+/34+/45+/AC133–)

Cell type-11

(AC133+)

Cell type-14

(CD34+/45+/AC133+)

Total Population, adjusted for ethnicity, hyperlipidemia, and cotinine n=210

β -1.783 -1.933 -0.047 -1.663

p-value 0.001* 0.001* 0.870 0.006*

Non-smokers,† adjusted for ethnicity, hyperlipidemia, and cotinine n=128

β -2.246 -2.274 -0.304 -1.945

p-value 0.004* 0.003* 0.364 0.001*

Caucasian, adjusted for hyperlipidemia, and cotinine n=118

β -1.788 -1.755 -0.410 -1.576

p-value 0.014* 0.017* 0.068 0.015*

African American, adjusted for gender, diuretics, calcium-channel blockers, SES, and cotinine n=88

β -1.418 -1.603 1.594 -7.106

p-value 0.044* 0.030* 0.013* 0.032*

Background Methods Results Discussion

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Figure 4A. Association between CVD risk and t,t-

MA. Mean t,t-MA levels for low (Framingham Risk

Score (FRS) < 20) and high (FRS ≥ 20 or

experienced a cardiovascular event) CVD risk

category were 0.14±0.03 (n=44) and 0.19±0.02

(n=166) mg/g creatinine, respectively.

Figure 4B. Association between CVD risk and t,t-

MA in non-smokers. Mean t,t-MA levels for low and

high CVD risk category in non-smokers (cotinine <

200 µg/g creatinine) were 0.10±0.03 (n=28) and

0.21±0.04 (n=98) mg/g creatinine, respectively.

Low High0.0

0.1

0.2

0.3

p = 0.024

CVD Risk Category (total population)

t,t-

MA

(m

g/g

cre

ati

nin

e)

A

Low High0.0

0.1

0.2

0.3p = 0.004

CVD Risk Category (non-smokers)

t,t-

MA

(m

g/g

cre

ati

nin

e)

B

Background Methods Results Discussion

Summary/ Discussion

1. t,t-MA levels were positively associated with cigarette smoke exposure.

2. Levels of cell types 2, 8, and 14 were inversely associated with t,t-MA levels. Benzene may increase CVD risk in part through the suppression of these cells.

3. t,t-MA levels were higher in people with high CVD risk regardless of smoking status.

4. Future research should determine which adverse CVD outcomes are linked with ambient air benzene exposures.

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Cell type-2, 8, and 14

Endothelial Dysfunction

BenzeneCVD

Air Pollution

Background Methods Results Discussion

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Discussion

Strengths

• Novel investigation of t,t-MA and CVD risk factors

• Large population study

• Individual measure of benzene exposure

• Empirical cigarette smoke exposure measurement

Limitations

• Biomarkers only collected at 1 time point

• Many other benzene metabolites

• Cotinine misclassification

• Possible selection bias

• No ambient benzene measurements

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Background Methods Results Discussion

Acknowledgements

• We thank the phlebotomists at the UofL Ambulatory Care and University Medical Associates for biological sample collection

• We thank Duane Bolanowski, Dave Young, Melissa Peak, Jordan Finch, the Jortani Laboratory, and the Diabetes & Obesity Center for technical assistance

• This work was supported in part by grants provided by the

• Anthem WellPoint Foundation (GMB090410)

• National Institute of Environmental Health Sciences (ES11860, ES019217)

• National Institute of General Medical Sciences (GM103492)

• Center for Environmental Genomics and Integrative Biology (P30ES014443)

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