characterization of endothelial cells from hereditary ... · endoglin expression in boecs eng...

Characterization of Endothelial cells from Hereditary Haemorrhagic Telangiectasia patients.

Africa Fernandez LopezCentro Investigaciones Biológicas

Madrid, Spain

Lab. Dr. Carmelo BernabéuDr. Luisa M. Botella

CONTROL HHT1 HHT2 n HHT2 m

Blood outgrowth endothelial cells characterization

Characterization

90% 2.3

PECAM-1 vW-FENDOGLIN ALK-1 VE-Cad F-Actin FLK1

- Controls- HHT1- HHT2 nonsense- HHT2 missense

CONTROL HHT2 nHHT1 HHT2 mHUVEC

Endoglin expression in BOECs

ENG response upon TGFβtreatment

0

0,5

1

1,5

2

2,5

2.5

1.5 1.5

1

Fold

indu

ctio

n

C

HHT1

HHT2

nHH

T2 m

0

500

1000

1500

2000

Expr

essi

onin

dex

0.5

1

C HHT1 HHT2n

0.6

HHT2m

0.3

ENG

β-Actin

C HHT1 HHT2n HHT2m

Endoglin expression byflow citometry

Endoglin expression

0

0,5

1

ALK-1KR

Fold

indu

ctio

n

0,8 ENG pXP2

pCDNA3 ALK1QD0

0,5

1,5

2

ALK-1WT

ALK1 acts over ENG promoter

**

*

Endoglin-(ZRP-1/Zyxin)-Actin interaction

Actin filament

ZyxinZRP1

ENG

(Conley et al. J Biol Chem. 2004)(Sanz-Rodriguez et al. J Biol Chem. 2004)

20 μm

Control

20 μm 20 μm

20 μm20 μm

HHT1

HHT2n HHT2m

Disorganized cytoskeleton in BOECs

Cytoskeleton disruption by siRNA

Control BOECs

pSUPER-Endo-Ex4 + pEGFP

Cytoskeleton organization recovery

20 μm

20 μm

20 μm

HHT1

HHT2m

20 μm20 μm20 μm

pCMV5-Endoglin + pEGFP

20 μm20 μm20 μm

(Fernandez-L et al. Cardiovasc Res. 2005)

Smad 2/3

ALK5 ALK1

Smad1/5

Proliferation

+-

ENG

+ENG

-

ALK1/ALK5 pathways equilibrium

Mutated ALK1/ENG

Do cells stop proliferating?

MEECsENG +/-

ALK5 (Lebrin et al., EMBO J., 2004)

ALK5

ENG ALK1

C HHT1 HHT2n HHT2m

GAPDH

ALK-5

RT-PCR

BOECs

01020304050

C HHT1 HHT2 n HHT2 m

Arb

itrar

yun

its

Densitometry

TGF-β

ALK5

ACTIVATION PHASERESOLUTION PHASE

ProliferationMigrationInvasion

ProliferationMigrationInvasion

Normal situation: Dynamic equilibrium

ALK1/ ENG

ALK1/ALK5 pathways equilibrium

Mutated ENG or ALK1: Unbalanced equilibrium

TGF-β

ALK5

ACTIVATION PHASERESOLUTION PHASE

ProliferationMigrationInvasion

ProliferationMigrationInvasion

ALK1/ ENG

Decreased ALK5 levels: New steady state

TGF-β

ALK5

ACTIVATION PHASERESOLUTION PHASE

ProliferationMigrationInvasion

ProliferationMigrationInvasion

ALK1/ ENG

ENG

ALK1 ALK5

Non-HHTENG

ALK1 ALK5

ENG

ALK1 ALK5

HHT-1 HHT-2

EndoglinALK-5

EndoglinALK-5

ALK-1 and ALK-5balanced

Altered angiogenesis

HHT PHENOTYPE

Altered ALK1/ALK5 signaling pathways

Decreased ENG levels

Weak cytoskeleton

Dr. Carmelo BernabéuDr. Luisa-María Botella

Dr. Francisco SanzFrancisco Javier BlancoÁfrica Fernández Eva María Garrido

Carmen Langa

This work has been partially supported by theHHT INTERNATIONAL FOUNDATION grant.