Angela S Benton, BAS 1 , Alan M Watson, PhD 1,2 , Zuyi Wang, PhD 1,3 ,

1. 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, DC 2The George Washington University, Washington, DC 3Virginia Polytechnic Institute and State University, Arlington, VA Delineation of a TIMP-1 Network Underlying the Response to Cigarette Smoke and Oxidative Stress in Asthma

2. Disclosure Statement The authors have documented that they have nothing to disclose.

3. 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)

4. 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)

5. Hypothesis Tobacco smoke exposure in asthmatic bronchial epithelium initiates an oxidative stress response Different from non-asthmatic bronchial epithelium

6. 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

7. Venn Diagram Analysis GSE3184 Lung tissue from HDM vs. PBS in vivo airway challenge in AJ and C3H mice GSE994 Epithelial brushings from smokers vs. nonsmokers N=2,034 N=130 N=29 N=238 N=8 1N=4 2N=6 N=367 N=236 GSE1301 Lung tissue from HDM vs. PBS in vivo airway challenge GSE3183 A549 cells in vitro treatment with IL13 vs. PBS 3N=2 N=3 N=99 4N=1 5N=17 N=9

8. Pathways Analysis Ingenuity Pathways Analysis™

9. 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)

10. 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

11. Coordinated Expression

12. Highly Regulated Genes

13. Fold Changes in Focus Network 0 hours 1 hour 8 hours 24 hours 72 hours

14. HBE Cell Donors • 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

15. In Vitro Validation A B *p=0.017 *p=0.011

16. 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

17. Conclusions TIMP-1 network Oxidative stress-induced pathway Underlies bronchial epithelial response to cigarette smoke and oxidative stress in asthma

18. New Questions • ETS-exposed children with asthma • Blunted TIMP-1 response? • More susceptible to metalloproteinase-mediated airway remodeling?

19. 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 • JinwookSeo, PhD • K12-RR core laboratories • NCMRR integrated molecular core laboratories (www.ncmrr.org) • GCRC genetics core laboratories • Genetic counseling • Microarray