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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