1. Endothelial Monocyte-Activating Polypeptide II (EMAP II) is a pro- inflammatory, anti-angiogenic and pro-apoptotic cytokine. Given its role in inflammation and apoptosis, key mechanisms in the pathophysiology of emphysema, we hypothesized that 1) EMAP II is a cigarette smoke-induced biomarker and mediator of emphysema, and 2) increases in lung EMAP II are sufficient to cause airspace enlargement. METHODS: C57/B16 and DBA2 mice were exposed to cigarette smoke (CS) for 4 months. Secreted lung EMAP II was neutralized with EMAP II antibody. Human BAL, serum and lung parenchyma from COPD patients were analyzed for EMAP II expression by immunoblotting and immunohistochemistry. RESULTS: CS exposure increased EMAP II expression in mouse and human lung. EMAP II antibody significantly inhibited the airspace enlargement induced by CS at 4 months. In humans, smoking and COPD were associated with increased EMAP II expression in the BAL, serum, and lung tissue. CONCLUSION: EMAP II expression is induced by CS exposure while EMAP II neutralizing antibody ameliorated CS-induced airspace enlargement. In humans, EMAP II expression was increased in active smokers and in those with emphysema. Our findings suggest that EMAP II may be a novel mediator and biomarker of cigarette-smoke induced emphysema. ABSTRACT BACKGROUND NOVEL ROLE OF EMAP II IN EMPHYSEMA N Sigua, G Rajashekhar, H Fehrenbach 2, K Kamocki, J Adamowicz, J Garrison, NI Rush, R Voswinckel 2, RM Tuder 3, HL Twigg III, M Clauss, I Petrache Indiana University, Indiana; 2 Univ of Marburg-Giessen, Germany; 3 UCHCS Denver, CO. Funding: R01 HL 077328 (IP) R01HL090950 (MC &IP) Inflammation, oxidative stress and alveolar endothelial apoptosis are central mechanisms in the pathophysiology of emphysema. Endothelial Monocyte-Activating Polypeptide II  pro-inflammatory, anti-angiogenic & pro- apoptotic cytokine  secreted in response to hypoxia, apoptosis and elastase/ caspase-3 activation  CXCR3 has been identified as a functional receptor  may be a molecule that causes both inflammation and apoptosis in emphysema  our previous work has shown that conditional lung-specific EMAP II over-expression utilizing a tetracyline- inducible construct under CCSP promoter causes increased airspace size and lung acinar volume, both changes consistent with emphysema.  C57/Bl6 and DBA2 mice were exposed to air control (AC) or cigarette smoke (CS) for 12 weeks. EMAP II neutralizing antibodies or isotype IgG control were administered via inhalation. EMAP II was conditionally overexpressed in the lung via a transgenic mouse model using a tetracycline inducible transactivator (TTA) controlled by the lung epithelium-specific CCSP promoter. ANIMAL EXPERIMENTS Fig 1: EMAP II expression in mouse broncho-alveolar lavage (a) and lung tissue (b) is increased by cigarette smoke (CS) exposure. Fig 2: EMAP II neutralization inhibited cigarette smoke-induced acinar airspace enlargement. Note the change in airspace size (a) and in the mean linear intercept (b), a measurement of alveolar diameter. HUMAN RESEARCH  52 volunteers were prospectively recruited from IU and Wishard clinics (IRB # 0709-76). Clinical data was gathered from retrospective chart review. EMAP II expression in serum was analyzed using immunoblotting.  Human BAL from smokers and nonsmokers from a separate IU cohort was tested for EMAP II expression using immunoblotting.  EMAP II expression was measured by immunohistochemistry of lung sections available from patients with COPD, idiopathic pulmonary fibrosis (IPF) and pulmonary arterial hypertension. Inclusion Criteria Age 18 and older AND one of the following:  Healthy non-smoking subjects  Active smokers with no active symptoms of COPD  Patients with established diagnosis of COPD Exclusion Criteria 1) Pregnancy; 2) COPD exacerbation defined as increase sputum production and purulence; 3) Acute illness which includes including fever, pneumonia, upper respiratory infection at the time or within 4 weeks prior to serum collection; 4) Volunteers who have other pulmonary diagnoses; 5) Volunteers with chronic liver disease, cancer or other systemic illnesses; and 6) Volunteers who are unable to give informed consent COPD Active smoker COPD Ex-smoker Healthy Active smoker Healthy Non- smoker Number (n)13 Mean Age (Years)53+1463+941+1944+19 Sex (Male/Female)4/97/60/134/9 Race (White/A.American)10/311/28/510/3 Smoking History (Packs/Year)46+4982+6918+160 Table 1: Characteristics of recruited subjects Fig 3: Increased pro-EMAP II expression in human serum in COPD smokers compared to healthy non-smokers. Fig 4: EMAP II expression in human bronchoalveolar lavage is increased in smokers compared to non- smokers. Fig 5: Increased EMAP II expression (immunohistochemistry) in patients with COPD/ emphysema vs. interstitial lung fibrosis or pulmonary arterial hypertension. REFERENCES: Barnett G, et al. Cancer Res 2000; Matschurat S, et al. Am J Pathol 2003;Hou Y, et al. Exp Hematol 2006.  Cigarette smoke exposure increased EMAP II expression.  EMAP II neutralization ameliorated CS-induced airspace enlargement in mice.  Active smoking and COPD increased EMAP II expression.  EMAP II may be a novel biomarker and mediator of cigarette-smoke induced emphysema.  We plan to develop improved quantitative assays to measure EMAP II in biological fluids and to further validate EMAP II as a potential diagnostic and therapeutic target in emphysema. CONCLUSION AND IMPLICATIONS