Medical School-University of Crete Head: Prof. NIKOLAOS SIAFAKAS MD
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1 LABORATORY OF MOLECULAR AND CELLULAR PULMONOLOGY Medical School-University of Crete Head: Prof. NIKOLAOS SIAFAKAS MD BACKGROUND The Department has published approximately 170 peer-reviewed papers on respiratory physiology and mechanics, diseases of the pleura, interstitial diseases, pharmacological clinical trials, quality of life, and molecular lung biology. Recently, we have detected instability of the microsatellite DNA (MI), and/or loss of heterozygosity (LOH) in sputum cells of patients with COPD, Asthma Allergic Rhinitis, and Interstitial Lung Diseases using highly polymorphic microsatellite markers. Furthermore, sputum induction (SI), bronchoalveolar lavage fluid (BALF), and lung biopsies are used to investigate different aspects of airway inflammation, angiogenic activity, oxidative stress, and apoptosis in COPD, Asthma and Interstitial Lung Diseases. RESEARCH 1. Somatic mutations and oxidative stress in COPD Previous studies suggested that somatic genetic alterations at the Microsatellite DNA may be of importance in COPD pathogenesis. We have shown that a significant proportion (49%) of COPD patients exhibited MSI in markers closely located to susceptibility genes involved in pathogenesis of the disease. Interestingly, COPD patients presented MSI in different chromosomal regions compared to asthmatics. Furthermore, MSI was found in sputum but not in nasal cytologic samples of COPD patients despite the fact that inflammation coexists in the nasal mucosa of these patients. Thus, MSI detection in COPD raises the questions when and how acquired somatic mutations at the microsatellite level occurring during the course of COPD, play a role in the pathogenesis or in the natural course of the disease. 142 We further attempted to investigate whether MSI is related to the exacerbation rate of COPD patients. Our results suggested that there is a potential relationship between an increased frequency of exacerbations and altered DNA repair process secondary to somatic mutations. A possible explanation for this association is that the increased airway inflammation and oxidative stress which are present in COPD have the potential to result in genetic alter-
2 LABORATORY OF MOLECULAR AND CELLULAR PULMONOLOGY ations or vice a versa. We are currently focusing our research on the relationship of somatic genetic alterations and oxidative stress in COPD patients, non-copd smokers and healthy subjects. 2. Surfactant protein-a (SP-A) in COPD. Pulmonary Surfactant proteins constitutes of a complex mixture of lipids and proteins that forms the mobile liquid phase covering the large surface area of the alveolar epithelium. It maintains minimal surface tension within the lungs in order to avoid lung collapse during respiration. Surfactant proteins also function in pulmonary host defence as immune mediators. Previous studies from our laboratory have detected Microsatellite DNA Instability (MSI) in sputum cells of patients with COPD, in D6S344 marker. This marker is located next to surfactant protein-a (SP-A) gene, and exhibited microsatellite DNA instability, only in COPD patients and not in non-copd smokers, or asthmatics. The expression levels of Surfactant protein in COPD and its association with the phenomenon of MSI could elucidate a part of the complex genetic basis of COPD. Currently, a study is in progress on the expression levels of SP-A protein in lung biopsies of COPD patients in comparison with non-copd smokers and healthy non-smokers. 3. Apoptosis and genetic instability in COPD. Recent data suggest that abnormal apoptotic events subvert cellular homeostasis and may play a primary role in the destruction of lung tissue, leading to emphysema. Especially in smok- ers who develop COPD there is an activation of adaptive immunity, with an infiltration of CD4 and primarily CD8 cells which are cytotoxic to epithelial cells through the release of granzymes and perforin, which can further induce apoptosis of alveolar cells.the objective of this project is to evaluate the expression levels of certain apoptotic markers in COPD and their association with the phenomenon of Microsatellite DNA Instability (MSI). The correlation of the genomic instability with apoptosis may shed light to COPD pathogenesis. 4. Pulmonary dendritic cells (DCs) in healthy non-smokers, healthy smokers and chronic obstructive pulmonary disease patients before and after smoking cessation. It has been hypothesized that DCs are central in the initiation and perpetuation of immune-mediated diseases, including asthma. Studies demonstrated a critical role for pul- monary DCs in allergic sensitization and pathogenesis of chronic inflammation in animal mod- 143
3 els of asthma. Much less is known about the role of DCs in COPD. In vitro studies have shown suppressive effects of cigarette-smoke on DCs. In vivo animal studies have yielded contradictory results, with other studies showing reduced DC numbers and maturation state after smoke exposure and others showing increased DC numbers and maturation state. We have recently shown that asthmatics who smoke have reduced bronchial mature DC numbers compared to healthy and asthmatic never-smokers. It is not known whether this reduction is due to cigarette smoking alone or to an interaction between smoking and asthma. We are currently investigated whether cigarette smoking decreases pulmonary DC numbers and their maturation state in healthy smokers and in COPD patients. 5. Expression analysis of CXCL12 and receptor CXCR4 in lung tissue and bone marrow mesenchymal stem cells in patients with idiopathic pulmonary fibrosis. The source of lung fibroblasts is a critical question in the pathogenesis of fibrotic lung diseases. It was suggested that fibrocytes (bone-marrow derived stem cells) could be the origin of these cells. It has been recently shown that the recruitment of fibrocytes to the lung is mediated via the interaction of the chemokine ligand, SDF-1/CXCL12 and its receptor, CXCR4. The aim of this study is to compare human lung tissue and bone marrow mesenchymal stem cells from patients with IPF to normal subjects for the expression of the biological axis CXCL12/CXCR4 in the pathogenesis of IPF. 6. Investigation of angiogenetic mechanisms in IPF, sarcoidosis and connective tissue disorders with interstitial lung involvement. The aim of this study is to evaluate the expression of cardinal growth factors (TGF- 1, CTGF, VEGF), chemokines (CXC angiogenic/ angiostatic), in order to identify alterations that maybe implicated in the pathogenesis of fibrotic lung disorders. 144
4 E 7. Pathophysiology of Obstructive Sleep Apneea Hypopnea Syndrome (OSAHS) and Cardiological consequences. OSAHS is a systemic rather than a local disease with familial predisposition and severe cardiological consequences. We currently focus our research to polyparametric analysis of OSAHS familial predisposition, studying several genes polymorphisms. In parallel inflammatory biomarkers evaluation is carried out related to vascular remodelling. REPRESENTATIVE PUBLICATIONS 1. NM Siafakas. In the Beginning of COPD. Is Evolution Important? Am J Respir Crit Care Med 2007;175: Tzortzaki EG, Lambiri I, Vlachaki E, Siafakas NM. Biomarkers in COPD. Curr Med Chem. 2007;14(9): Tsoumakidou M, Elston W, Zhu J, Wang Z, Gamble E, Siafakas NM, Barnes NC, Jeffery PK. Cigarette smoking alters bronchial mucosal immunity in asthma. Am J Respir Crit Care Med. 2007;175(9): Antoniou KM, Tzouvelekis A, Alexandrakis MG, Sfiridaki K, Tsiligianni I, Rachiotis G, Tzanakis N, Bouros D, Milic-Emili J, Siafakas NM. Different angiogenic activity in pulmonary sarcoidosis and idiopathic pulmonary fibrosis. Chest Oct;130(4): Zervou MI, Tzortzaki EG, Makris D, Gaga M, Zervas E, Economidou E, Tsoumakidou M, Tzanakis N, Milic-Emili J, Siafakas NM. Differences in microsatellite DNA level between asthma and chronic obstructive pulmonary disease. Eur Respir J. 2006;28(3): Simantirakis EN, Schiza SE, Chrysostomakis SI, Chlouverakis GI, Klapsinos NC, Siafakas NM, Vardas PE. Atrial overdrive pacing for the obstructive sleep apnea-hypopnea syndrome. N Engl J Med. 2005;353(24): GROUP MEMBERS Prof. Nikolaos Siafakas MD, PhD (Head) Nikolaos Tzanakis MD, PhD (Group Leader) Eleni Tzortzaki MD, PhD (Group Leader) Sophia Schiza MD, PhD (Group Leader) Katerina Antoniou, MD, PhD Irini Lambiri, MD, PhD Maria Tsoumakidou MD, PhD Maria Plataki MD, PhD Dimitris Vassilakis MD, PhD Maria Zervou MSc, PhD Elina Vlachaki MD, (PhD student) Foteini Economidou MD, (PhD student) Katerina Samara MD, (PhD student) Izolde Bouloukaki MD, (PhD student) 145
5 Christina Thomou MD, (PhD student) Marianna Siganaki (PhD student) Irini Neofytou, MSc (PhD student) Ioannis Politis (PhD student) George Margaritopoulos (PhD student) FUNDING European Commission FP5 program BIOAIR European Commission FP6 program GALEN EPEAEK PYTHAGORAS II 2004 Glaxo-SmithKline Research Grants 2003, 2004, 2005, 2006 Hellenic Thoracic Society Research Grant 2006 COLLABORATORS Prof. Sven-Erik Dahlen, Karolinska Institute, Stockholm, Sweden Prof. Eleni Papadaki, Medical School, University of Crete Dr. Anastassios Koutsopoulos, Medical School, University of Crete Dr. Emmanuel Paraskakis, Medical School, University of Crete Dr. Demosthenes Makris, Medical School, of University of Thessaly, Larissa. CONTACT Eleni Tzortzaki, MD, PhD Lecturer in Thoracic Medicine University Hospital, Medical School University of Crete, Heraklion 71110, Crete, Greece. Tel: Fax:
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