CEC, EMP, EPC - GFTC : Groupe Francophone thrombose et cancer

Cellules endothéliales circulantes
Progéniteurs et microparticules endothéliales:
Vers la définition de la vasculo-compétence
Janvier 2011
F. DIGNAT-GEORGE
UMR 608 INSERM - Université de la Méditerranée
« Physiopathologie de l’ Endothélium »
Marseille
Plasticity of endothelial phenotype
Biomechanical stimuli
Fluid shear stress, hydrostatic pressure, Cyclic strain………
Blood-born
mediators
Cells
blood and
neighboring tissues
Cytokines , Thrombin,
Bacterial products…
Physiological
adaptation
VASCULAR
HOMEOSTASIS
from a quiescent
to an activated phenotype
Pathophysiological
dysfunction
VASCULAR
DISORDERS
How to explore vascular endothelium ?
The ideal test: safe, non invasive, easy to perform, cheap and standardized
INVASIVE
NON INVASIVE
Biopsy
Surrogate biomarkers
Morphology
Soluble markers
Immuno-histology
- hemostasis (vWF, sTF, sTM, t-PA, …)
- adhesion ( sVCAM, sE-selectin,…)
- angiogenesis(VEGF , bFGF, HGF…)
Cellular markers
CEC
EMP
EPC
Functional
testing
Endothelium dependent
vasorelaxation (FMD)
Arterial stiffness
Pulse wave velocity
Spectrum of endothelial responses to injury
Activation
Dysfunction / Injury
Regeneration
SMC
CEC
Pathogenic factors
(CVRF …)
EC
Procoagulant and
Proinflammatory
Activation
EPC
EMP
vesiculation
detachement
repair
signalisation
ECM
Endothelial integrity : a balance
MP from TNF-stimulated HUVEC
MP from normal plasma
VASCULAR COMPETENCE
EMP, CEC, EPC: Key players of the vascular homeostatic balance
Emergent biomarkers integrated into an endothelial phenotype
reflecting the injury/repair equilibrium and
defining « vascular competence » at the individual level
From the bench to the bedside
International Society on Thrombosis and
Haemostasis
Scientific and Standardization Committee (SSC)
in vascular biology
F. Dignat-George
http://www.isth2010.com/
Pre‐analytical and analytical steps Questions
Novel insights provided by CEC , EMP and EPC
as biomarkers of endothelial injury/ repair equilibrium
• Rapid overview focusing on their definition,
measurement and major clinical significance
• Relevance to study endothelial alterations in vascular
disorders
• Integrative approach to define vascular competence
How to define CEC ?
ISTH: Proposal of a consensus definition of CEC
Blann AD, Woywodt A and Dignat-George F, Thromb Haemost, 228-35, 2005
• Mature EC shed from the vessel wall
• Reflective of endothelial damage or death
• Nucleated cells of 15-50µm diameter
• Expression of endothelial markers :
CD146, vWF, CD31…
• Lack of expression of leukocyte (CD45)
and immaturity markers (CD133)
• No clonogenic potential
vWF+
• Rare Events
Consensus protocol for CEC enumeration
(Woywodt A Blann AD and Dignat-George F, J Thromb Haemost. 2006 Mar;4(3):671-7)
Numeration and identification by fluorescence
microscopy
Identification criteria
- Rosettes bearing more than 5 beads
- Size higher than 15 µm
- Coexpression of endothelial markers
Normal values
Below 10 CEC/ml
New hybrid assays for CEC detection
First step : Pre-enrichment of CD146+ cells using paramagnetic
particles coated with SEndo-1 mAb (ferrofluides), and immunostaining
Second step : Multiparametric identification
- Flow cytometry
CellQuant FF-CD146®
Widemann et al, J Thromb haemost, 2008
- Image analysis
Cellsearch ®
Jason Rowand et al, Cytometry, 2007
Normal values : below 20 CEC/ml
Imaging analysis
CEC significance in vascular diseases
Biomarkers of endothelial lesion
• Diagnostic value
• Correlation with disease activity and prognosis
significance
• Treatment monitoring
Pathogenic vectors
- Dissemination of a TF dept procoagulant potential
- Amplification of a pro inflammatory potential
(Mutin et al, Blood , 1999)
(Woywodt et al, Blood , 2008)
Diagnostic value of CEC in vascular disorders
Combined use of CEC and cardiac troponin improves diagnosis
accuracy in patients with UA
Quilici et al, Circulation, 2005
50
Troponin-, CEC40
13
Number of
patients
10
30
13
Troponin-, CEC+
1
3
Troponin+, CEC+
9
12
6
20
Troponin+, CEC-
2
24
10
14
16
0
H0
H4
Time after admission
H8
Diagnostic value of CEC in vascular disorders
Circulating Endothelial Cells : a new candidate biomarkers of irreversible
pulmonary hypertension secondary to congenital heart disease
Smadja et al, Circulation 2009
R.PAH
I.PAH
Bcl-2
VEGF
CEC counts are increased in CHD patients
with irreversible pulmonary hypertension
Histological changes in pulmonary arteries
identify a « proliferative apoptosis
resistant phenotype
Pronostic value of CEC in vascular disorders
Elevation of CEC independently predicts cardiovascular events and mortality.
Hemodialysis
Koc M et al, Kidney Int 2005,
Acute coronary syndromes
Boos CJ. Eur Heart J. 2007 ;28(9):1092-101.
Value of CEC for Therapeutic monitoring
Bonello L et al, JACC 2010
CEC changes evidences protective effect of efficient clopidogrel therapy on
endothelium in patients undergoing percutaneous coronary intervention
Patients were stratified according to their responsiveness to
clopidogrel defined by VASP index
« Isolation of putative progenitor endothelial
cells for angiogenesis »
(Asahara et al, Science, 1997; 275: 964-7)
Human adult peripheral blood
Immuno Magnetic Selection of CD34+ / VEGF-R2+ cells
Endothelial
Progenitor Cells
In vitro culture
Proliferation and differentiation
into cells with functional and
phenotypic properties of
endothelial cells
Injection in animal
models
Incorporation into
angiogenic sites
Promotion of endothelial
repair/growth
Heterogeneity of EPC
(adapted from Leri et al, Circ Res, 2005)
Others sources
Hémangioblast
Bone
marrow
?
HPSC
MAPC
MSC
?
?
Hematopoïetic progenitors Endothelial progenitors
Vessel wall, Heart, liver, adipose tissue
Peripheral blood
Monocytes
« myeloïd sub type »
« Early EPC »
EPC
« true angioblasts »
« Late EPC »
Contribution to neovascularisation/vascular repair
Phenotype of EPC ?
CEC
EPC
CD34/KDR
HPC
Ashara et al, Science 97
CD34
KDR
CD146
CD144
CD45neg
CD146/CD31
CD133 ?
Peichev, Blood 2000
Case, Exp hematol 2007
Estes, Cytometry 2010
CD45 dim ?/ neg ?
Timmermans, ATVB 2007
Estes, Curr Proc Cytom 2010
Need for discriminative marker
CD34
CD133
CD45 dim
How to explore EPC ?
• Numeration
Peripheral blood
34/KDR
- phenotypic identification (FCM)
a technical « challenge »
- Identification based on clonogenic activity
« Endothelial CFU assays »
Heterogeneous approaches
• Fonctional studies
- Endothelial differentiation
- Proliferation, Migration
- Adhesion
- tubes formation
- Incorporation into endothelial monolayer
-…
CFU-EC
EPC significance in vascular diseases
Increased EPC levels are associated
• to acute ischemia or vascular injury
Myocardial ischemia, unstable angina, stroke, coronary stent implantation ….
mobilization correlates with favorable outcome
• Pathological angiogenesis
Malignancies
Rheumatoid Arthritis
Proliferative retinopathy
EPC activity correlates with disease severity
EPC significance in vascular diseases
Decreased EPC levels
- Are associated to cardiovascular diseases
(Coronary artery disease, in-stent restenosis, cerebrovascular disease, heart
failure, pulmonary hypertension …)
- Are indicative of cumulative cardiovascular risk factors
(Diabetes, age, chronic renal insufficiency, smoking, dyslipidemia …)
- Inversely correlate with endothelial dysfunction
- Identify patients with poor cardiovascular outcome
Low EPC levels identify patients at increased
cardiovascular risk
Werner N. et al, N Engl J Med 2005
519 patients with CAD, follow-up : 1 year
Baseline levels of EPC
Cumulative free survival rate
Major Cardiovascular Events
Death from cardiovascular causes
EPC constitute a protective ongoing vascular repair able to:
- counterbalance CVRF impact
- modulate the clinical course of atherosclerosis
- determine cardiovascular outcome
Characterisation of EMP
Combes et al, J Clin Invest, 1999
• Vesicles resulting from blebbing of the cell membrane in response to cell
activation or apoptosis
• Heterogeneous diameter (0.1 to 1 µm)
• Phosphatidylserine exposure and endothelial marker expression
EMP generation in response
to TNF stimulation
CONTROL
EMP: Miniature version of endothelial cells
PECAM-1
TF
TM
TNF
EPCR
uPAR
VCAM-1
RNA
MMP
ICAM-1
Intracellular
protein
E-selectin
uPA
FcR
MHC
Multifaceted endothelial microparticles:
A storage pool of bioactive vectors
at the crossroad of thrombosis, inflammation and angiogenesis
F Dignat-George et al, ATVB, 2011 ,Sabatier et al, J Cell Mol Med, 2009
r
100 nm
Vascular remodeling – angiogenesis
IL-1alpha production Y Berda- G Kaplanski
Proteases
EMP
IInflammation
Combes et al,
J.Clin Invest 1999
Coagulation
Sabatier et al, Blood, 2002
Monocytes
(uPA/uPAr, MMP)
Lacroix et al, Blood, 2007
Jouvencel , Lacroix R et al, Blood 2010
TF
TF
TNF
LPS
Thrombus formation
Microparticle determination
Flow cytometry
VE cadherin, Endoglin, E selectin,CD146
Numeration
Patent : n° 9908505
Fluorescent Calibrated beads ( Biocytex)
(Robert et al, J.Thromb. Haemost. 2008)
( Lacroix et al; J Thromb Haemost,2009)
Functional assays
PS /Prothrombinase activity
TF/ Thrombin generation
Procoagulant potential
tPA: uPA / plasmin activity
Patent : n° 07/04060
Proteolytic activity
Captured assays ( JM Freyssinet / F Toti)
Endothelial antigen/prothrombinase activity
Procoagulant potential / Cellular origin
EMP significance in vascular diseases
Sabatier et al, J Cell Mol Med, 2009
•
Biomarkers of endothelial activation/injury
Correlation with disease activity and with endothelial dysfunction
Vasculitis
( Brogan et al, Arth Rheum, 2004)
Coronary artery disease
( Werner N et al, 2006)
End stage renal disease
(Amabile N et al, 2006)
Sickle cell anemia
•
Met syndrome
Renal failure
(Chironi et al , Hypert 2009)
(Sabatier et al, Diabetes 2002
Chahed et al, Diabetes 2009)
(Helal, Nutrition and Metabolism, 2010)
(Jourde et al, J. Throm. Haemost., 2009
Faure et al, J Throm. Haemost., 2006)
Pronostic significance, vascular risk
Stroke
Type 2 diabetes mellitus
Patients at risk for CHD
•
( Shet AS, Blood, 2003 )
Hypertension
Diabetes
Lupus anticoagulant
(Combes , J. Clin. Invest. 2001) AntiphospholipidSyndrome (Dignat‐George , J. Throm. H. 2003
(Koga et al, 2005,Morel 2007)
Bed rest
Sabatier , Am J Physiol, 2010)
(Nozaki et al, 2009)
Kydney transplantation (Al Massarani Am. J. Transpl. 2008)
(Simak et al 2006)
Treatment monitoring
Calcium inhibitors
Pioglitazone
Eicosapentanoic acid
Transplantation
Insulinothérapie
(Nomura et al, 2007)
(Esposito et al, ATVB 2006 )
( Morel et al, Thromb Haemost, 2004
(Massarani Am. J. Transpl. 2009)
(Darmont et al, Thromb. Research 2010)
Pathogenic vectors
Bioactive vectors disseminating procoagulant ,proinflammatory and proteolytic activites in the bloo
Endothelial MP levels are significantly higher
in patients with Chronic Renal Failure
Role of uremic toxins ?
p<0.01
30
p<0.01
20
Actin
cytoskeleton
Control
10
pc 10µg/ml pc 50µg/ml
Cerini et al, Thromb Haemost 2005
p<0.0001
1500
0
Controls
CRF
HD
EMP levels remained significantly increased
even after exclusion of diabetic patients
EMP/103 HUVEC cells
CD144+ EMP/µL of plasma
VE-cadherin
p<0.0001
1000
500
0
Hcy
Faure V et al , J Thromb Haemost, 2006
indoxyl
sulfate
p-cresol
oxalate
Cellular origin of MP and vascular dysfunction in CRF
Amabile N, JASN 2005.
Elevated MP of endothelial, platelet and erythrocyte origin
in 44 dialyzed patients with end-stage renal failure
In vivo
EMP correlate with
impaired vascular function
Brachial artery
Flow-mediated
dilation
(flow-mediated dilation , aortic pulse wave velocity)
In vitro
EMP impair NO production by
endothelial cells
(rat aortic rings model)
Impairment of relaxation to acetylcholine
L-NAME+/- MPs
EMP ESRF
CD144 MPs
EMP healthy
EMP are highly associated with arterial dysfunction
Effect of renal transplantation on endothelial biomarkers
Al-Massarani et al, Am J Transplant. 2008
Patients (n=52)
CEC/mL
(mean ± SEM)
EMP/µL
(mean ± SEM)
sVCAM-1 (ng/mL)
(mean ± SEM)
Before graft
One year post
graft
Healthy control
donors
(n=50)
43.6± 7.9*
24 ± 4.1*#
4.6 ± 0.63
15.1 ± 1.8*
7.7 ± 2.2#
9.9 ± 1.2
1495 ± 107*
659 ± 57*#
449 ± 18
*: significant relative to HCD # : significant relative to before graft
No significant correlation was found between CEC, EMP and sVCAM-1 levels
either before or after graft.
No correlation with age, gender, underlying kidney disease,
Impact of previous history of cardiovascular
diseases on CEC, EMP, and sVCAM-1 levels
before and one year after graft
-History of cardiovascular disease ( n=11) : myocardial infarction, coronary heart disease,
stroke, and symptomatic peripheral vascular disease
-Patients w and wo HCVD were similar with regards to classical biological risk factors
before and one year after renal transplantation
* p< 0.05 vs before graft in patients without HCVD, ** p<0.05 for HCVD vs w/o HCVD at M12
Significant decreased levels of CEC and EMP one year post graft,
only in patients wo HCVD
Although renal transplantation improves endothelial alterations, some of the cardiovascular risk
factors may still exert deleterious effect in the post-transplant period
Questions
Novel insights provided by CEC , EMP and EPC
as biomarkers of endothelial injury/ repair equilibrium
• Rapid overview focusing on their definition,
measurement and major clinical significance
• Relevance to study endothelial alterations in vascular
disorders
• Integrative approach to define vascular competence
Insight from endothelial biomarker profile
1. Identification of pathogenic mechanism contributing to
endothelial dysfunction: adaptative response to injury
50 children with Systemic Vasculitis
Clarke LA et al, Athritis Rheum 2010
CEC
EMP
EPC
- Increased levels of endothelial injury
- Increased EPC levels
vasculogenic response in children
as a marker of putative adaptation to injury
Insight from endothelial biomarker profile
2/ Identification of pathogenic mechanism contributing to
endothelial dysfunction/ Lack of adaptative response to injury
• EMP and EPC in stable CAD patients with or
without preserved left ventricular function
EPC positively correlated with FMD
Bulut et al, J Cardiac Fail 2008
EMP inversely correlated with FMD
Increased endothelial damage and lack of compensatory mechanisms
contribute to pathologic development of endothelial dysfunction
Insight from endothelial biomarker profile :
3/ Improved sensibility for evaluation of endothelial targeting
therapy
Damage Index : Apoptotic CEC/EPC
**
**
Apoptotic CEC/EPC :
43 patients with CAD
Group A: de novo Atrovastatin 40/mg/d ;
group B : Ezetimibe 10mg/d
group C : Atorvastatin dose escalation
(4w therapy)
Surrogate index for evaluation of the
pleitropic effects of statins
Schimdt-Lucke C et al , Atheroscl 2010
Towards the definiton of vascular competence
MP from TNF-stimulated HUVEC
MP from normal plasma
VASCULAR COMPETENCE
Vascular Competence Index
Pattern of endothelial biomarkers
Altered profile
Normal profile
CEC-EMP
vascular competence
competence.
Vascular
PEC
Deleterious
Stimuli
VCI
Adaptative response toward the
reestablishment of vascular homeostasis
Altered profile
Low levels of CEC, EMP, EPC :
Physiological quiescence of
endothelium
Vascular incompetence
Inadapted response to pathological processes
that compromises endothelium status
Conclusion
¾ CEC, EMP, EPC
Emerging markers of endothelial injury or regeneration
¾ Standardization of methodologies
A crucial step in the full definition of their clinical value
¾ Endothelial biomarker profile
Extented vision of endothelial dynamics
¾ Vascular competence index
Identification of cardiovascular risk
Definition of therapeutic strategies
Acknowledgements
UMR INSERM U608 : Physiopathologie de l’endothélium
Laboratoire d’Hématologie et d’Immunologie
F. Sabatier
R. Lacroix
J. Sampol
L. Camoin-Jau
P Paul
L. Dou
P. Brunet
N. Jourde
C. Cerini
S. Robert
L . Arnaud
EuroCECnetwork
A Blann
A Woywodt
………….
Clinical Dept
Marseille
JL Bonnet
J Quilici
F. Paganelli
L Bonello
JR Harle
Y Berland
ISTH SSC
Vascular Biology
JM Freyssinet
N Key
French EPC
Network
(INSERM)
P. Gaussem
G Uzan
R & D coll.
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