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