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Angelo Avogaro
Università degli Studi di Padova
La carenza di cellule endoteliali progenitrici:un passaggio chiave nell’ambito della patologia
cardiovascolare del malato di diabete.
Universa Universis Patavina Libertas
Fibrinolysis Platelets aggregation Coagulation Permeability Adhesiveness Infiammation
PAI-1t-PA NO
PGI2
TXA2 Fibrinogen
Thrombomodulin
vWF
TFRAGE
MCP-1
P-selectin
ICAM-1
VCAM-1
NO
PGI2
EDHF TXA2 ET-1
VEGF
PDGF TGF-β
VasodilatationVasodilatation VasocostrictionVasocostriction ProliferationProliferation
Causes of endothelial dysfunction in Diabetes
1. Impaired
ROS
ROS
PKCPolyolsAGEHexosamines
L-arginine L-citrulline
NOS3
NONO
ADMABH4
Vasodilatation Vasodilatation
ROSGlycated proteins
Glucose Glucose
NAD(P)H Oxidase
NAD(P)H Oxidase
Changes in Coronary segment diameters expressed as percentage of baseline in response to Acetylcholine
(Adapted from Nitenberg et al. Diabetes 1993)
P<0.01 P<0.001 P<0.001 P<0.001 n.sC vs. D
Postprandial Myocardial Perfusionis impaired in Type 2 Diabetic Patients
(Scognamiglio et al. 2005)
Baseline Postprandial
0
5
10
15
Myo
card
ial B
lood
Flo
w
Changes in Coronary segment diameters expressed as percentage of baseline in response to Acetylcholine
(Adapted from Nitenberg et al. Diabetes 1993)
P<0.01 P<0.001 P<0.001 P<0.001 n.sC vs. D
Circulating Endothelial Cells in Cardiovascular Disease (Boos et al. 2006)
Blood VesselCEC
CD 133+CD 146-
RFRF
Endothelial Precursor Cells: a novel approach to assess vascular integrity
• 0.1% to 3.0% of endothelial cells proliferate daily
• Endothelial cell division may reach 50% of the cells in and around the injured sites
Ingram et al. Blood 2005
Circulating endothelial cells are elevated in patients with type 2 diabetes mellitus independently of HbA1c
(McClung et al. 2005)
Lamalice, L. et al. Circ Res 2007;100:782-794
Major steps of endothelial cell migration
Risk factors
Apoptosis
Apoptosis Regeneration
EPC
Endothelial cell apoptosis and -regeneration
Bone Marrow ↔ Circulating Progenitor Cells Bone Marrow ↔ Circulating Progenitor Cells
Bone marrow cellsBone marrow cells Peripheral blood
CD34+ cell pool
Endothelial progenitor cells
(KDR+)
Cardiomyocyte progenitor cells
(c-met+/CXCR4+)
Smooth muscle progenitor cells
(-actin+)
Apoptosis
Other CD34+ derived phenotypes
EPCsEPCs
HSCs
Injured endothelium
Repair
VSMCs migration
From BONE MARROW
New vessel growth
Differentiation
Differentiation
??
IschemiaVEGFVEGFSDF-1SDF-1PlGFPlGFFGFFGFEPOEPO
Defective mobilizationDecreased survivalIncreased homing
Deranged differentiation
Low EPCs
Mechanisms of Progenitor Cell Decrease
Fadini et al. Curr Diabetes Rev 2005
0
100
200
300
400
500
600
700
800
0 5000 10000
BM CD34+ cells
PB
CD
34
+ c
ells
.
0
100
200
300
400
500
600
700
800
0 5000 10000
BM CD34+ cells
PB
CD
34
+ c
ells
.
Bone marrow and peripheral blood progenitor cells
r = 0.51p = 0.017
r = 0.51p = 0.017
Bone marrow and peripheral blood sampled during open heart surgeryBone marrow and peripheral blood sampled during open heart surgery
Diabetes Mellitus and EPCs Diabetes Mellitus and EPCs (Tepper et al. Circulation 2002; Loomans et al. Diabetes 2004; Fadini et al. JACC 2005)(Tepper et al. Circulation 2002; Loomans et al. Diabetes 2004; Fadini et al. JACC 2005)
Diabetes Mellitus and EPCs Diabetes Mellitus and EPCs (Tepper et al. Circulation 2002; Loomans et al. Diabetes 2004; Fadini et al. JACC 2005)(Tepper et al. Circulation 2002; Loomans et al. Diabetes 2004; Fadini et al. JACC 2005)
0
50
100
150
200
250
300
350
400
450
CD34+ CD34+KDR+
Pro
ge
nit
or
Ce
lls
.
CTRL DM 2
0
50
100
150
200
250
300
350
400
450
CD34+ CD34+KDR+
Pro
ge
nit
or
Ce
lls
.
CTRL DM 2
0
10
20
30
40
50
60
70
80
90
100
Control DM2
EP
C c
ou
nt
.
0
10
20
30
40
50
60
70
80
90
100
Control DM2
EP
C c
ou
nt
.
CTRLCTRL DM2DM2
Cultu
reCu
lture
Adhe
sion
Adhe
sion
HbA1c (%)HbA1c (%)
EPC
coun
tEP
C co
unt
0
1
2
3
4
5
6
7
8
9
10
Control DM1
EP
C c
ou
nt
.
0
1
2
3
4
5
6
7
8
9
10
Control DM1
EP
C c
ou
nt
.
HbA1c (%)HbA1c (%)
EPC
coun
tEP
C co
unt
Krankel, N. et al. Arterioscler Thromb Vasc Biol 2005;25:698-703
The quantitative analysis revealed a significant decrease in the CPC amount after culture in HG medium (A)
The clinical significance of EPCs(Fadini et al. ATVB 2006)
The clinical significance of EPCs(Fadini et al. ATVB 2006)
0102030405060708090
100
0 1 2 3 4 5 6
CD
34
+K
DR
+ c
ells
.
0102030405060708090
100
0 1 2 3 4 5 6
CD
34
+K
DR
+ c
ells
.
0
20
40
60
80
100
<30 30-50 51-70 >70
CD
34
+K
DR
+ c
ells
.
0
20
40
60
80
100
<30 30-50 51-70 >70
CD
34
+K
DR
+ c
ells
.
PAD: Disease Stage of Rutheford CCA plaque obstruction
0
10
20
30
40
50
60
70
80
90
CTRL IGT IFG DMC
D3
4+
KD
R+
ce
lls
.
*
0
10
20
30
40
50
60
70
80
90
CTRL IGT IFG DMC
D3
4+
KD
R+
ce
lls
.
*
050
100150200250300350400450500
CTRL IGT IFG DM
CD
34
+ c
ell
s
.
**
050
100150200250300350400450500
CTRL IGT IFG DM
CD
34
+ c
ell
s
.
**
The clinical significance of EPCsThe clinical significance of EPCsDisease Marker – Prediabetes Disease Marker – Prediabetes (Fadini et al Diabetologia 2007)(Fadini et al Diabetologia 2007)
The clinical significance of EPCsThe clinical significance of EPCsDisease Marker – Prediabetes Disease Marker – Prediabetes (Fadini et al Diabetologia 2007)(Fadini et al Diabetologia 2007)
Reduced survivalReduced survival
012345
6789
10
CT HG HG+BFT MAN
CFU
-EC
/ fie
ld
.
012345
6789
10
CT HG HG+BFT MAN
CFU
-EC
/ fie
ld
.
0
0,2
0,4
0,6
0,8
1
1,2
CT HG HG+BFT MAN
pFO
XO
3A
.
0
0,2
0,4
0,6
0,8
1
1,2
CT HG HG+BFT MAN
pFO
XO
3A
.
Federici et al. Diabetes 2006Federici et al. Diabetes 2006
Glucose (mM)Glucose (mM)
50
60
70
80
90
100
110
120
0 5 10 15 25 50 25
EPC
/ hi
gh p
ower
fiel
d
.
50
60
70
80
90
100
110
120
0 5 10 15 25 50 25
EPC
/ hi
gh p
ower
fiel
d
.
Seeger et al. Circulation 2005Seeger et al. Circulation 2005
****
********
****
GlucoseGlucoseP38 P38 inhibinhib -- -- ++GlucoseGlucoseP38 P38 inhibinhib -- -- ++
5050
6060
7070
8080
9090
100100
110110
120120
00 2525 2525
EPC
/ hi
gh p
ower
fiel
d
.
EPC
/ hi
gh p
ower
fiel
d
.
5050
6060
7070
8080
9090
100100
110110
120120
00 2525 2525
EPC
/ hi
gh p
ower
fiel
d
.
EPC
/ hi
gh p
ower
fiel
d
.
Impaired mobilization Impaired mobilization (Fadini et al. Diabetologia 2006)
0
50
100
150
200
250
300
350
400
Basal d1 d3 d7
Sc
a-1
+ c
-kit
+ c
ells
.
0
50
100
150
200
250
300
350
400
Basal d1 d3 d7
Sc
a-1
+ c
-kit
+ c
ells
.
I/R injuryI/R injury
ControlDiabetesDiabetes + insulin
ControlDiabetesDiabetes + insulin 0
100
200
300
400
500
600
700
0 250 500 750 1000 1250 1500 1750
Plasma SDF-1 (pg/mL)
Sc
a1
+ c
-Kit
+ c
ells
.
r = 0.85*
r = 0.72*
0
100
200
300
400
500
600
700
0 250 500 750 1000 1250 1500 1750
Plasma SDF-1 (pg/mL)
Sc
a1
+ c
-Kit
+ c
ells
.
r = 0.85*
r = 0.72*
0
1000
2000
3000
4000
5000
6000
7000
8000
Sc
a-1
+ c
ell
s
. CTRL DM
0
50
100
150
200
250
Basal G-CSF+SCF I/R 2h/72h
sc
a-1
+ c
-kit
+ c
ell
s
.
0
1000
2000
3000
4000
5000
6000
7000
8000
Sc
a-1
+ c
ell
s
. CTRL DM
0
50
100
150
200
250
Basal G-CSF+SCF I/R 2h/72h
sc
a-1
+ c
-kit
+ c
ell
s
.
G-CSF 50 G-CSF 50 mg/kgmg/kg
+ SCF 200 + SCF 200 mg/kgmg/kg5 days5 days
adjustedobservedadjustedobserved
Progressive progenitor cell decline
N = 425N = 425
Adjusted for age, sex, plasma glucose, HbA1c, BMI, smoke, blood pressure, lipid profile, diabetic complications and CVD, medicationsAdjusted for age, sex, plasma glucose, HbA1c, BMI, smoke, blood pressure, lipid profile, diabetic complications and CVD, medications
-300
-250
-200
-150
-100
-50
0
50
100
150
200NGT IFG IGT
DMnew
DM<10
DM 10-20
DM>20
CD
34+
Cel
l co
un
t
CD34+ progenitor cells metabolic componentsCD34+ progenitor cells metabolic components
0
50
100
150
200
250
300
350
400
450
500
None One Two Three Four Five
Components of the Metabolic Syndrome
CD
34
ce
ll c
ou
nt
.
Predicted Observed
< 3 components
= 3 components: diagnosis of MS
*
*
0
50
100
150
200
250
300
350
400
450
500
None One Two Three Four Five
Components of the Metabolic Syndrome
CD
34
ce
ll c
ou
nt
.
Predicted Observed
< 3 components
= 3 components: diagnosis of MS
*
*
Synergistic reduction of CD34+ progenitors at clustering cardiometabolic risk factors
Correlation between CD34+ cells and HOMA, a measure of insulin resistance
Synergistic reduction of CD34+ progenitors at clustering cardiometabolic risk factors
Correlation between CD34+ cells and HOMA, a measure of insulin resistance
Fadini et al. Eur Heart J 2006Fadini et al. Eur Heart J 2006
0
2
4
6
8
10
12
no SMET SMET
Ha
zard
Ra
tio
High CD34+ cell countLow CD34+ cell count
0
2
4
6
8
10
12
no SMET SMET
Ha
zard
Ra
tio
High CD34+ cell countLow CD34+ cell count
0
5
10
15
20
25
30
no SMET SMET
Ha
zard
Ra
tio
High CD34+ cell countLow CD34+ cell count
All events All events CV events CV events Death Death
Progenitor Cells and Outcomes in MetSyn Patients
After correction for age, sex, lipid profile, blood pressure, family After correction for age, sex, lipid profile, blood pressure, family history, smoking habit, obesity, CRP, plasma glucose, renal function, history, smoking habit, obesity, CRP, plasma glucose, renal function, baseline CVD, metabolic syndrome, 10-yr Italian risk score.baseline CVD, metabolic syndrome, 10-yr Italian risk score.
****
n = 214n = 214
Atherosclerosis in press
The bone marrow connectionThe bone marrow connection
Bone marrow defectBone marrow defect
Endothelial progenitor cellsEndothelial progenitor cells
Cardiomyocyte progenitor cellsCardiomyocyte progenitor cells
Smooth muscle progenitor cellsSmooth muscle progenitor cells
CD34+Sca-1+c-kit+
CARDIOVASCULAR COMPLICATIONSCARDIOVASCULAR COMPLICATIONS
DIABETES MELLITUSDIABETES MELLITUS
AcknowledgmentsAcknowledgmentsAcknowledgmentsAcknowledgments
PADOVA EPC Study Group
METABOLIC DIVISION Gianpaolo FadiniSaula de Kreutzenberg
GENERAL PATHOLOGYSaverio SartoreMattia AlbieroStefano Schiaffino
CLINICAL IMMUNOLOGYCarlo AgostiniElisa Boscaro
Partly supported by the Heart Repair consortium
PADOVA EPC Study Group
METABOLIC DIVISION Gianpaolo FadiniSaula de Kreutzenberg
GENERAL PATHOLOGYSaverio SartoreMattia AlbieroStefano Schiaffino
CLINICAL IMMUNOLOGYCarlo AgostiniElisa Boscaro
Partly supported by the Heart Repair consortium