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.