1. Methods Human bronchial epithelial cells (16HBE) were stimulated for 24 hrs, 120 hrs and 12 days with 5% cigarette smoke extract (CSE), to evaluate Oct-4 and CD146 protein expression by Western Blot analysis and Flow cytometry, as previously described (4). Immunoreactivity for Oct-4 and CD146 was assessed in bronchial biopsies obtained from COPD (n=11; GOLD stages I-III) and HC (n=6) by immunohistochemistry. Statistical comparisons in order to test differences between the two groups (COPD and HC) were made by use of the Mann Whitney U Test. ANOVA test was used for the analysis of the data obtained from in vitro experimental conditions. * p<0.05; ** p<0.01. Bibliography 1.Hogg JC, Timens W. The pathology of chronic obstructive pulmonary diseases.. Ann Rev Pathol 2009. 4:435-459. 2.Buitrago W, Roop DR. Oct-4: the almighty POUripotent regulator?. J Invest Dermatol. 2007;127(2):260-2. 3.Ouhtit A, Gaur RL, Abd Elmageed ZY, Fernando A, Thouta R, Trappey AK, Abdraboh ME, El-Sayyad HI, Rao P, Raj MG. Towards understanding the mode of action of the multifaceted cell adhesion receptor CD146. Biochim Biophys Acta. 2009;1795(2):130-6. 4.Brigham C. Willis, Zea Borok. TGF-β-induced EMT: mechanisms and implications for fibrotic lung disease. Am J Physiol Lung Cell Mol Physiol 2007;293:L525-34 5.Gagliardo R Chanez P et al. IκB kinase-driven nuclear factor-κB activation in patients with asthma and chronic obstructive pulmonary disease J All Clin Immunol 2011.128(3):635-45. Role of chronic exposure to cigarette smoke on Oct-4/CD146 imbalance in human bronchial epithelial cells. R. Gagliardo, F. Bucchieri, A.M. Montalbano, R. Marchese, F. Rappa, P. Tralongo, G.D. Albano, G. Anzalone, G. Paglino, M. Profita. Institute of Biomedicine and Molecular Immunology, Italian National Research Council, Palermo, Italy Department of Experimental Biomedicine and Clinical Neurosciences, University of Palermo, Palermo, Italy Centro Oncologico di III Livello La Maddalena, U.O. di Pneumologia Interventistica, Italy. Abstract The airway epithelium is a dynamic tissue that undergoes slow but constant renewal. Dysregulation of airway epithelial cell function related to cigarette smoke exposure plays an important role in the pathophysiology of COPD and is associated to tissue damage and disease severity (1). Oct-4 is the crucial POU domain transcription factor responsible for maintaining cellular self-renewal and regeneration (2), and CD146 is an adhesion molecule involved in outside-in signalling of inflammatory processes, remodeling and cell proliferation (3). The aim of the present study was to investigate the mechanisms involved in airway epithelium maintenance and injury-induced regeneration and their correlation with Oct-4 and CD146 protein expression. We studied the expression of Oct-4 and CD146 protein by western blot and flow cytometry in human bronchial epithelial cells (16HBE cells) exposed to 5% Cigarette Smoke Extract (CSE) for 24 hrs, 120 hours and 12 days. The epithelial immunoreactivity for Oct-4 and CD146 was also assessed in bronchial biopsies from COPD and healthy control subjects (HC). We found that long term exposure to CSE significantly down-regulated Oct-4 protein expression in 16HBE cells, whereas CD146 was significantly increased by CSE treatment. The Oct-4 immunoreactivity in bronchial epithelium was significantly reduced in COPD in comparison to HC, whilst CD146 expression was higher in COPD than in HC. Chronic exposure to cigarette smoke may be able to induce an altered epithelium homeostasis and regenerative capacity of bronchial epithelial cells. The Oct-4 gene activity down-regulation might lead to the imbalance between renewal, repair processes and pro-inflammatory and pro-oxidative mechanisms with a potential role in abnormal tissue remodeling and progression of COPD. Figure #1Figure #2 Objectives To evaluate mechanisms involved in repair and regeneration of lung epithelium. To address the role of oxidative stress induced by cigarette smoke exposure in epithelium homeostasis and regenerative capacity. To determine mechanisms involved in an altered epithelium regeneration and inappropriate tissue repair. Results Conclusions Our results showed that long term exposure to CSE significantly down-regulated Oct-4 protein expression in 16HBE cells, whereas CD146 was significantly increased by CSE treatment. The immunoreactivity for Oct-4 in bronchial epithelium was significantly reduced in COPD than in HC, whilst CD146 expression was higher in COPD than in HC. Oxidative stress-mediated mechanisms sustain the in vitro and ex vivo down-regulation of the transcription factor Oct-4 and the induction of the cell adhesion receptor CD146. The imbalance between Oct-4 and CD146 might promote an altered epithelium regeneration and inappropriate tissue repair, leading to tissue remodeling and progression of COPD. Flow cytometry for CD146 expression. The effects of 5%CSE on CD146 receptor expression was evaluated by flow cytometry in 16HBE cells after 24hrs, 120 hrs and 12 days. Effects of long term exposure to 5%CSE on Oct-4 and CD146 protein expression in 16HBE cells by Western Blot analysis. A) and B) Oct-4 and CD146 protein expression in presence or absence of 5% CSE. c) Representative Western Blotting of Oct-4 and CD146 protein expression in presence or absence of 5% CSE. Immunohistochemistry for Oct-4 and CD146 in bioptic samples from COPD and Healthy Control subjects. A) and B) Expression of Oct-4 and CD146 proteins in bronchial biopsies isolated from COPD and HC subjects. C) Representative immunostaining for Oct-4 and CD146. Figure #3 Background The chronic inflammatory insult promotes a progressive damage of the entire lung. The balance between inflammatory processes and repair mechanisms is actually considered a crucial phenomenon in the fate of pathological processes. A high tissue destruction in the absence of appropriate repair mechanisms leads to permanent structural alterations. It is hypothesized that abnormal epithelial damage caused by inflammation and oxidative stress may reactivate the EMTU, resulting in excessive reparative response by the epithelium and a consequent “remodeling” of the airways. The term “remodeling” in fact refers to the disruption of the normal architecture of tissue, in response to an ineffective reparative reaction. COPD is characterized by abnormal signals occurring between the epithelium, that is in contact with the outside world, and the underlying cell populations (in particular dendritic cells and myofibroblasts), aimed at reactivating the EMTU, thus determining the “remodeling” (4). Oct-4 is the crucial POU domain transcription factor responsible for maintaining cellular self-renewal and regeneration, and CD146 is an adhesion molecule involved in outside-in signalling of inflammatory processes, remodeling and cell proliferation. 0 20 40 60 80 100 120 Epithelium cells /mm Oct-4 CD146 Negative controlCOPDHC 0 25 50 75 100 125 150 175 200 225 Epithelium cells /mm COPD HC COPDHC ** Baseline 5% CSE 0 5 10 15 20 25 24 hrs120 hrs15 days * ** CD146 (% gated) Baseline 15 days 100 101 102 103 104 CD146 Baseline 24 hrs 5% CSE 24 hrs 5% CSE 120 hrs 5% CSE 15 days CD146 Baseline 120 hrs 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 Oct-4 CD146 0,2,4,6,8 1 A) ** 24 hrs120 hrs12 days Oct-4 expression (arbitrary units) ** 0,2,4,6,8 1 ** 24 hrs120 hrs12 days CD146 expression (arbitrary units) B) Baseline 5% CSE 16HBE CD146  -actin 72 kDa 42 kDa Oct-4 39 kDa 24 hrs120 hrs12 days A) B) C) B) A) C)