LABORATORY OF MOLECULAR AND CELLULAR PULMONOLOGYMedical 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 chromo-

somal 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.

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 inflamma-

tion and oxidative stress which are present in COPD have the potential to result in genetic alter-

LABORATORY OF MOLECULAR AND CELLULAR PULMONOLOGY

142

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 sub-

jects.

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 res-

piration. 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 surfac-

tant 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 pro-

tein 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-

LABORATORY OF MOLECULAR AND CELLULAR PULMONOLOGY

143

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 contradic-

tory 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 investigat-

ed 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 dis-

eases. 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 mediat-

ed 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 connec-

tive 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.

LABORATORY OF MOLECULAR AND CELLULAR PULMONOLOGY

144

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 inflamma-

tory 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;175:1-2.

2. Tzortzaki EG, Lambiri I, Vlachaki E, Siafakas NM. Biomarkers in COPD.

Curr Med Chem. 2007;14(9):1037-48

3. 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):919-25.

4. 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. 2006

Oct;130(4):982-8.

5. 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):472-8.

6. Simantirakis EN, Schiza SE, Chrysostomakis SI, Chlouverakis GI, Klapsinos NC,

Siafakas NM, Vardas PE. Atrial overdrive pacing for the obstructive sleep apnea-hypop-

nea syndrome. N Engl J Med. 2005;353(24):2568-77.

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)

LABORATORY OF MOLECULAR AND CELLULAR PULMONOLOGYE

145

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: +30 2810 392 433

Fax: +30 2810 542 650e-mail: tzortzaki@med.uoc.gr

LABORATORY OF MOLECULAR AND CELLULAR PULMONOLOGY

146