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

Disclosure Statement The authors have documented that

they have nothing to disclose.

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)

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)

Hypothesis Tobacco smoke exposure in

asthmatic bronchial epithelium initiates an oxidative stress response Different from non-asthmatic bronchial

epithelium

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

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

Pathways Analysis

Inge

nuity

Pat

hway

s Ana

lysis

™

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)

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

Coordinated Expression

Highly Regulated Genes

Fold Changes in Focus Network

1 hour

8 hours

24 hours

72 hours

0 hours

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

In Vitro Validation

*p=0.017 *p=0.011

A B

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

Conclusions TIMP-1 network

Oxidative stress-induced pathway Underlies bronchial epithelial response to

cigarette smoke and oxidative stress in asthma

New Questions ETS-exposed children with asthma

Blunted TIMP-1 response? More susceptible to metalloproteinase-

mediated airway remodeling?

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