Angela S Benton, BAS 1 , Alan M Watson, PhD 1,2 , Zuyi Wang, PhD 1,3 ,
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Transcript of Angela S Benton, BAS 1 , Alan M Watson, PhD 1,2 , Zuyi Wang, PhD 1,3 ,
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Angela S Benton, BAS1, Alan M Watson, PhD1,2, Zuyi Wang, PhD1,3,Mary C Rose, PhD1,2, and Robert J Freishtat, MD, MPH1,2 1Children's National Medical Center, Washington, DC2The George Washington University, Washington, DC3Virginia Polytechnic Institute and State University, Arlington, VA
Delineation of a TIMP-1 Network Underlying the Response to Cigarette Smoke and Oxidative Stress in Asthma
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Disclosure Statement The authors have documented that
they have nothing to disclose.
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Tobacco Smoke in Childhood Asthma Asthma = syndrome with genetic and
environmental components (FD Martinez, 1997, 2007)
Tobacco smoke Common environmental trigger Associated with diagnosis and increased
morbidity(Y Chen et al, 2005; J Cunningham et al, 1996; FD Gilliland et al, 2006; MK Selgrade et al, 2006; JJ Sturm et al, 2004)
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Tobacco Smoke Effects onBronchial Epithelial Cigarette smoke
Respiratory epithelial response Mediated in part by oxidative stress Asthmatic bronchial epithelium more
susceptible to this stress (Bucchieri et al, 2002)
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Hypothesis Tobacco smoke exposure in
asthmatic bronchial epithelium initiates an oxidative stress response Different from non-asthmatic bronchial
epithelium
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Data Integration Four publicly-available data series
NCBI: Gene Expression Omnibus (GEO) Asthma- and cigarette smoke-relevant N = 153 arrays Unsupervised clustering Adequate signal/noise levels Genespring GX10
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Venn Diagram Analysis
1N=4
GSE1301Lung tissue from HDM vs. PBS in vivo airway challenge
N=238 N=8
3N=2 N=99N=3
N=9
2N=6
N=29
GSE3183A549 cells
in vitro treatment with IL13 vs. PBS
N=367
GSE3184Lung tissue from HDM vs. PBS in vivo airway challenge in AJ and C3H mice
N=2,034
N=236
GSE994Epithelial brushings
from smokers vs. nonsmokers
N=130
4N=1 5N=17
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Pathways Analysis
Inge
nuity
Pat
hway
s Ana
lysis
™
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Tissue Inhibitor of Metalloproteinase (TIMP) -1 Secreted 31 kDA glycoprotein Inhibits protease activity of all MMPs (DE Gomez et
al, 1997) MMP-9 major lung MMP (WC Parks and SD Shapiro, 2001; H
Tanaka et al, 2000) Asthmatic BALF and sputum levels lower than
control(W Mattos et al, 2002; AM Vignola et al, 1998)
MMP-9:TIMP-1 hypothesized to drive extracellular lung remodeling (JJ Atkinson et al, 2003)
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Phosgene-Induced Oxidative Stress Mice exposed to phosgene at time 0 Sacrificed at eight post-exposure time
points (0.5, 1, 4, 8, 12, 24, 48, 72 hours) Lung tissue isolated RNA extracted Whole genome expression profiling
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Coordinated Expression
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Highly Regulated Genes
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Fold Changes in Focus Network
1 hour
8 hours
24 hours
72 hours
0 hours
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Primary human differentiated respiratory epithelium (HBE) 1 hour exposure: cigarette smoke
condensate (CSC) or hydrogen peroxide 23 hour: incubation ELISA: measure protein levels of TIMP-1 and
MMP-9 in the apical and basal secretions
HBE Cell Donors
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In Vitro Validation
*p=0.017 *p=0.011
A B
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Summary 4-way Venn diagram approach
Publicly-available microarray data Identified TIMP-1 nucleated network of
proteins Key in response to tobacco smoke and
oxidative stress in asthma In vitro validation
Potential for subepithelial conditions known to favor airway remodeling in chronic asthma
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Conclusions TIMP-1 network
Oxidative stress-induced pathway Underlies bronchial epithelial response to
cigarette smoke and oxidative stress in asthma
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New Questions ETS-exposed children with asthma
Blunted TIMP-1 response? More susceptible to metalloproteinase-
mediated airway remodeling?
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Acknowledgements Research Center
for Genetic Medicine @ Children’s National Medical Center Freishtat Lab
K23-RR-020069 Rose Lab
Mary Rose, PhD Alan Watson, PhD
Hoffman Lab Eric Hoffman, PhD Zuyi Wang, PhD Jinwook Seo, PhD K12-RR core laboratories NCMRR integrated
molecular core laboratories (www.ncmrr.org)
GCRC genetics core laboratories Genetic counseling Microarray