ABSTRACT

1. NOVEL ROLE OF EMAP II IN EMPHYSEMA N Sigua, G Rajashekhar, H Fehrenbach2, K Kamocki, J Adamowicz, J Garrison, NI Rush, R Voswinckel2, RM Tuder3, HL Twigg III, M Clauss, I Petrache Indiana University, Indiana; 2Univ of Marburg-Giessen, Germany; 3UCHCS Denver, CO. Funding: R01 HL 077328 (IP) R01HL090950 (MC &IP) ABSTRACT ANIMAL EXPERIMENTS HUMAN RESEARCH 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. • 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. • 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. Fig 4: EMAP II expression in human bronchoalveolar lavage is increased in smokers compared to non-smokers. • 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 Fig 5: Increased EMAP II expression (immunohistochemistry) in patients with COPD/ emphysema vs. interstitial lung fibrosis or pulmonary arterial hypertension. BACKGROUND • 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. Fig 1: EMAP II expression in mouse broncho-alveolar lavage (a) and lung tissue (b) is increased by cigarette smoke (CS) exposure. CONCLUSION AND IMPLICATIONS • 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. Table 1: Characteristics of recruited subjects 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. REFERENCES: Barnett G, et al. Cancer Res 2000; Matschurat S, et al. Am J Pathol 2003;Hou Y, et al. Exp Hematol 2006. Fig 3: Increased pro-EMAP II expression in human serum in COPD smokers compared to healthy non-smokers.