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,
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
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
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
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.
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
Table 1: Demographic and spirometric characteristics of IPF patients
Number 10 10
Sex: Male/Female 5/5 7/3
Age, median (yr) 59 (32- 65) 65(40-75)
6/ 4 8/10
FVC, (% pred) 103 + 14 77.3+ 13.0*
TLC,( % pred) 101 + 19 67.4+14.2*
(% pred) 96 + 6 60.3+17.8*
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
Capacity for Carbon Monoxide; PαO2
, Arterial Partial Pressure of Oxygen
MSC characterization was based on morphology,
immunophenotypic profile (CD34-, CD45-, CD14-,
CD90+, CD105+, CD73+, CD146+) and differentiation
potential towards three lineages
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).
1. MSC culture and identification1. MSC culture and identification
Immunophenotypic characteristics ofImmunophenotypic characteristics of
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
Antoniou KM, Papadaki HA, Soufla G, et al. ERS 2008
Antoniou KM, Papadaki HA, Soufla G, et al. ERS 2008
MSCs IPF patients (n=10) and age-/sex-matched healthy
individuals (n=10) were similar in frequency,
differentiation potential, immunophenotypic
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
1. Either the increased expression of
CXCR4 occurred as a result of lung injury
2. it preceded lung injury
We furthermore investigated telomere length in
BM-MSCs and telomerase expression in lung
tissue of patients with IPF
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
Two components: hTERT, hTRTwo components: hTERT, hTR
Telomerase activity is clearly required for cancer cell
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
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
•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
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 tissue
Antoniou KM, et al. AJRCCM 2009
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.
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
Dept of Haematology
Prof Helen Papadaki
Christina Kastrinaki, PhD
Dept of Clinical Virology
Prof DA Spandidos
G. Sourvinos, As. Prof