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  1. 1. Thank you for viewing this presentation. We would like to remind you that this material is the property of the author. It is provided to you by the ERS for your personal use only, as submitted by the author. © 2008 by the author
  2. 2. Increased CXCR4 expression and short telomeres in bone marrow mesenchymal stem cells of patients with idiopathic pulmonary fibrosis Foteini Economidou, Katerina M. Antoniou, Giannoula Soufla, Athanasia Proklou, Rena Lymbouridou, Helen Papadaki and Nikolaos M. Siafakas Departments of Thoracic Medicine, Virology and Haematology, Medical School, University of Crete, Heraklion, Greece
  3. 3.  Idiopathic Pulmonary fibrosis (IPF) is a devastating condition that leads to progressive lung destruction and scarring.  The mean survival time following diagnosis is less than 5 years.  No effective treatment  Unknown pathogenesis
  4. 4. IntroductionIntroduction  The source of fibroblasts involved in the pathogenesis of fibroticThe source of fibroblasts involved in the pathogenesis of fibrotic lung disorders is unknown.lung disorders is unknown.  Recent evidence indicates that a significant proportion ofRecent evidence indicates that a significant proportion of mesenchymal cells (MSCs) involved in this repair/remodelingmesenchymal cells (MSCs) involved in this repair/remodeling process may be derived from extrapulmonary sources, such as theprocess may be derived from extrapulmonary sources, such as the peripheral blood (fibrocytes) and the bone marrow (progenitorperipheral blood (fibrocytes) and the bone marrow (progenitor cells).cells).  It has been also suggested that the recruitment of MSCs to the lungIt has been also suggested that the recruitment of MSCs to the lung is mediated via the interaction of the biological axisis mediated via the interaction of the biological axis CXCL12/CXCR4.CXCL12/CXCR4.  This hypothesis is supported further by experimental evidence thatThis hypothesis is supported further by experimental evidence that antibody neutralization of CXCR12 reduces recruitment ofantibody neutralization of CXCR12 reduces recruitment of fibrocytes and pulmonary fibrosis.fibrocytes and pulmonary fibrosis.  In contrast, other studies suggest a protective effect of the boneIn contrast, other studies suggest a protective effect of the bone marrow-derived cells.marrow-derived cells.
  5. 5. From Dunsmore S and Shapiro S, JCI 2004
  6. 6. Strieter RM, et al. JCI 2007
  7. 7. Aim of the studyAim of the study  This study investigates:This study investigates:  the reserves and function, the molecular andthe reserves and function, the molecular and proteomic profile of BM MSCs andproteomic profile of BM MSCs and  the expression of the biological axisthe expression of the biological axis CXCL12/CXCR4 in patients with IdiopathicCXCL12/CXCR4 in patients with Idiopathic Pulmonary Fibrosis (IPF) in comparison withPulmonary Fibrosis (IPF) in comparison with healthy controlshealthy controls..  to probe the possible involvement of MSCs in the pathogenesis of IPF
  8. 8. Table 1: Demographic and spirometric characteristics of IPF patients Characteristics Control subjects IPF patients Number 10 10 Sex: Male/Female 5/5 7/3 Age, median (yr) 59 (32- 65) 65(40-75) Smokers/non smokers 6/ 4 8/10 FVC, (% pred) 103 + 14 77.3+ 13.0* TLC,( % pred) 101 + 19 67.4+14.2* TLCO, (% pred) 96 + 6 60.3+17.8* PαO2, ( mmHg) - 80.3+10.0 Values are expressed as mean + SD, and age as median (range). * Statistically significance difference between IPF patients and healthy controls (p<0.05). Abbreviations: FVC, Forced Vital Capacity; TLC, Total Lung Capacity; TLCO Diffusing Capacity for Carbon Monoxide; PαO2 , Arterial Partial Pressure of Oxygen
  9. 9. Methods (I)  MSC characterization was based on morphology, immunophenotypic profile (CD34-, CD45-, CD14-, CD90+, CD105+, CD73+, CD146+) and differentiation potential towards three lineages (adipocytes/chondrocytes/osteocytes). Kastrinaki MC, et al. Ann Rheum Dis 2007  The frequency of MSCs in the BM mononuclear cell fraction was evaluated by using a limiting dilution assay.  We have also assessed the molecular and proteomic characteristics in terms of inflammatory cytokine gene and protein expression of BM MSCs (VEGF, TGF-β1, FGF-b and SDF/ CXCR4).
  10. 10. Methods (II) 1. MSC culture and identification1. MSC culture and identification  Immunophenotypic characteristics ofImmunophenotypic characteristics of MSCs.MSCs.  Trypsinized MSCs from passage-2 (P2) wereTrypsinized MSCs from passage-2 (P2) were immunophenotypically characterised by flow cytometryimmunophenotypically characterised by flow cytometry  Differentiation potential of MSCs at P2  Telomerase activity and telomere length 2. Real-time PCR 3. ELISA for protein evaluation
  11. 11. by Papadaki HA
  12. 12. Antoniou KM, Papadaki HA, Soufla G, et al. ERS 2008
  13. 13. Antoniou KM, Papadaki HA, Soufla G, et al. ERS 2008
  14. 14. Results  MSCs IPF patients (n=10) and age-/sex-matched healthy individuals (n=10) were similar in frequency, differentiation potential, immunophenotypic characteristics.  A significant increase in the mRNA expression has been detected in both SDF-1 –TR1 (mean + SD, 1502+ 4180 versus 36+ 28, p=0.002) and CXCR4 (median, 34325 versus 1.54, p=0.002) in IPF patients.  No statistical difference was found in TGF-β1, FGF-b and VEGF mRNA expression levels between patients and controls.
  15. 15. Discussion  1. Either the increased expression of CXCR4 occurred as a result of lung injury or  2. it preceded lung injury
  16. 16.  We furthermore investigated telomere length in BM-MSCs and telomerase expression in lung tissue of patients with IPF
  17. 17. Telomerase  A specialized ribonucleoprotein-containing enzyme  synthesizes telomere DNA to prevent degeneration of chromosomal ends in actively dividing cells.  Telomerase adds telomere repeats to ends ofTelomerase adds telomere repeats to ends of chromosomeschromosomes  Two components: hTERT, hTRTwo components: hTERT, hTR  Telomerase activity is clearly required for cancer cell propagation  Its role in the injured lung is unknown
  18. 18. Telomeres and IPF  Telomeres are DNA-protein structures that protect chromosome ends.  Short telomeres activate a DNA damage response that leads to cell death or permanent cell cycle arrest.  Telomere length predicts the onset of various diseases.
  19. 19. Telomere length in familial IPF  Specific mutations identified in a fewSpecific mutations identified in a few family casesfamily cases  Early onset of the disease was related toEarly onset of the disease was related to specific telomerase mutationsspecific telomerase mutations Armanios et al NEJM 2007 Tsakiri et al PNAS 2007
  20. 20. •25% of sporadic cases and 37% of familial cases of pulmonary fibrosis had telomere lengths <10th percentile •This cannot be explained by coding mutations in telomerase. •Telomere shortening of circulating leukocytes may be a marker for an increased predisposition toward the development of this age-associated disease. Cronkhite JT et al. AJRCCM 2008
  21. 21. •Telomere length as determined by the quantitative PCR assay for normal controls (green triangles) and IPF samples (blue spots) plotted against age. Antoniou KM, Papadaki HA, Soufla G, Siafakas NM. AJRCCM 2009 (letter)
  22. 22. Telomerase activity in lung tissue Antoniou KM, et al. AJRCCM 2009
  23. 23. ConclusionsConclusions The mobilisation and the reduction ofreduction of telomerase length in BM-MSCs may suggest atelomerase length in BM-MSCs may suggest a pathogenetic role of those cells in IPF.pathogenetic role of those cells in IPF.
  24. 24. Dept of Thoracic Medicine Head: Professor Nikolaos Siafakas Interstitial Lung Disease Group Katerina Antoniou, Lecturer Foteini Economidou, MD, PhD Giorgos Margaritopoulos, MD Athanasia Proklou, MD Giannoula Soufla, PhD Rena Lymbouridou, PhD student Konstantinos Karagiannis, PhD student Ismini Lasithiotaki Collaborators: Dept of Haematology Prof Helen Papadaki Christina Kastrinaki, PhD Helen Koutala Athina Damianaki Dept of Clinical Virology Prof DA Spandidos G. Sourvinos, As. Prof

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