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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
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
IntroductionIntroduction The source of fibroblasts involved in the pathogenesis of fibrotic The source of fibroblasts involved in the pathogenesis of fibrotic
lung disorders is unknown. lung disorders is unknown.
Recent evidence indicates that a significant proportion of Recent evidence indicates that a significant proportion of mesenchymal cells (MSCs) involved in this repair/remodeling mesenchymal cells (MSCs) involved in this repair/remodeling process may be derived from extrapulmonary sources, such as the process may be derived from extrapulmonary sources, such as the peripheral blood (fibrocytes) and the bone marrow (progenitor peripheral blood (fibrocytes) and the bone marrow (progenitor cells). cells).
It has been also suggested that the recruitment of MSCs to the lung It has been also suggested that the recruitment of MSCs to the lung is mediated via the interaction of the biological axis is mediated via the interaction of the biological axis CXCL12/CXCR4. CXCL12/CXCR4.
This hypothesis is supported further by experimental evidence that This hypothesis is supported further by experimental evidence that antibody neutralization of CXCR12 reduces recruitment of antibody neutralization of CXCR12 reduces recruitment of fibrocytes and pulmonary fibrosis. fibrocytes and pulmonary fibrosis.
In contrast, other studies suggest a protective effect of the bone In contrast, other studies suggest a protective effect of the bone marrow-derived cells.marrow-derived cells.
From Dunsmore S and Shapiro S, JCI 2004
Strieter RM, et al. JCI 2007
Aim of the studyAim of the study
This study investigates:This study investigates: the reserves and function, the molecular and the reserves and function, the molecular and
proteomic profile of BM MSCs andproteomic profile of BM MSCs and the expression of the biological axis the expression of the biological axis
CXCL12/CXCR4 in patients with Idiopathic CXCL12/CXCR4 in patients with Idiopathic Pulmonary Fibrosis (IPF) in comparison with Pulmonary Fibrosis (IPF) in comparison with healthy controlshealthy controls. .
to probe the possible involvement of MSCs in the pathogenesis of IPF
Table 1: Demographic and spirometric characteristics of IPF patients
CharacteristicsControl 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
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).
Methods (II)
1. MSC culture and identification1. MSC culture and identification Immunophenotypic characteristics of Immunophenotypic characteristics of
MSCs.MSCs. Trypsinized MSCs from passage-2 (P2) were Trypsinized MSCs from passage-2 (P2) were
immunophenotypically characterised by flow cytometry immunophenotypically characterised by flow cytometry Differentiation potential of MSCs at P2 Telomerase activity and telomere length
2. Real-time PCR3. ELISA for protein evaluation
by Papadaki HA
Antoniou KM, Papadaki HA, Soufla G, et al. ERS 2008
Antoniou KM, Papadaki HA, Soufla G, et al. ERS 2008
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.
Discussion
1. Either the increased expression of CXCR4 occurred as a result of lung injury or
2. it preceded lung injury
We furthermore investigated telomere length in BM-MSCs and telomerase expression in lung tissue of patients with IPF
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 of chromosomesTelomerase adds telomere repeats to ends of chromosomes
Two components: hTERT, hTRTwo components: hTERT, hTR
Telomerase activity is clearly required for cancer cell propagation
Its role in the injured lung is unknown
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.
Telomere length in familial IPF
Specific mutations identified in a few Specific mutations identified in a few family casesfamily cases
Early onset of the disease was related to Early onset of the disease was related to specific telomerase mutationsspecific telomerase mutations
Armanios et al NEJM 2007Tsakiri et al PNAS 2007
•25% of sporadic cases and 37% of familial cases of pulmonary fibrosis hadtelomere 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
•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)
Telomerase activity in lung tissueAntoniou KM, et al. AJRCCM 2009
ConclusionsConclusions
The mobilisation and the reduction of reduction of telomerase length in BM-MSCs may suggest a telomerase length in BM-MSCs may suggest a pathogenetic role of those cells in IPF. pathogenetic role of those cells in IPF.
Dept of Thoracic MedicineHead: Professor Nikolaos Siafakas
Interstitial Lung Disease GroupKaterina Antoniou, Lecturer
Foteini Economidou, MD, PhDGiorgos Margaritopoulos, MDAthanasia Proklou, MDGiannoula Soufla, PhDRena Lymbouridou, PhD studentKonstantinos Karagiannis, PhD studentIsmini Lasithiotaki
Collaborators:
Dept of Haematology
Prof Helen PapadakiChristina Kastrinaki, PhDHelen KoutalaAthina Damianaki
Dept of Clinical Virology
Prof DA SpandidosG. Sourvinos, As. Prof