Endoglin has a crucial role in blood cell-mediated

vascular repair

Linda van Laake

NIOB

Heart Lung Center Utrecht, Cardio-Thoracic Surgery / Cardiology

Netherlands Institute for Developmental Biology (Hubrecht Laboratory)

NIH workshop – HHT Bethesda, 8th June 2006

Background

• Endoglin: accessory receptor for TGF-β in vascular endothelial cells (EC)– Essential for angiogenesis (mouse

development)• Hereditary hemorrhagic telangiectasia

type 1 (HHT1): mutations endoglin gene– Vascular malformations increasing with age– Haploinsufficiency– Clinical manifestations variable

Background

• Mononuclear cells (MNCs) can express endoglin– Endothelial progenitor cells, circulating EC,

bone marrow monocytic lineages• MNCs contribute to vascular repair

– Transdifferentiation to EC; vasculogenesis– Secretion growth factors and cytokines;

angiogenesis

Hypothesis

• Vascular repair, mediated by MNCs, may be impaired in subjects with HHT1

• Model– HHT1 patients and mice for endoglin mutation– Myocardial infarction (MI) for angiogenesis

and vasculogenesis

MI in mice

• Myocardial infarction or

• Sham thoracotomy

• 4 week survival >60%

• Endoglin heterozygous mice (Eng+/-)• Wildtype littermates (Eng+/+)• Wildtype Balb/C

MNC injection

• Venous blood from HHT1 patients or healthy volunteers

MNC injection

• Venous blood from HHT1 patients or healthy volunteers

• Density gradient centrifugation MNC population

• 1-3 hours after MI 5 million cells (or PBS) in tail vein

• Tacrolimus (Prograft®) for immunosuppression

Analysis

• Endoglin expression infarcted vs. healthy heart (human and mouse): ISH and IHC

Analysis

• Endoglin expression infarcted vs. healthy heart (human and mouse): ISH and IHC

• Homing human MNC to infarct : cryosections – UEA-1 lectin+FITC (4d)

Analysis

• Endoglin expression infarcted vs. healthy heart (human and mouse): ISH and IHC

• Homing human MNC to infarct : cryosections – UEA-1 lectin+FITC (4d)

• Vessel and inflammatory cell count: PECAM (1w, 2w, 4w), CD45, CD68, Mac-3 (1w)

Analysis

• Endoglin expression infarcted vs. healthy heart (human and mouse): ISH and IHC

• Homing human MNC to infarct : cryosections – UEA-1 lectin+FITC (4d)

• Vessel and inflammatory cell count: PECAM (1w, 2w, 4w), CD45, CD68, Mac-3 (1w)

• Heart function: mouse MRI (1w, 4w)

Magnetic Resonance Imaging

4 Chamber

Short axis

Ejection Fraction (EF)= (EDD-ESD) / EDD

Cardiac index = EF*EDD*heart rate / weight

End Diastolic Diameter (EDD)

End Systolic Diameter (ESD)

Analysis

• Endoglin expression infarcted vs. healthy heart (human and mouse): ISH and IHC

• Homing human MNC to infarct : cryosections – UEA-1 lectin+FITC (4d)

• Vessel and inflammatory cell count: PECAM (1w, 2w, 4w), CD45, CD68, Mac-3 (1w)

• Heart function: mouse MRI (1w, 4w)• Statistical analysis: Mann-Whitney U test

Endoglin expression

Azan endoglinmRNA

0.5 mm 0.5 mm

0.5 mm

50 µm

PECAM endoglinprotein

50 µm

50 µm 50 µm

Healthy

Infarct

• Endoglin upregulated in neoangiogenicvessels formed after MI (human and mouse)

50 µm

50 µm

50 µm

50 µm

Endoglin and neovascularization

PECAM endoglin

Eng+/+

Eng+/-

• Reduced upregulation of endoglin and neoangiogenesis in Eng+/- mice after MI

• CD45, CD68, Mac-3: no difference

Non-infarctedmyocardium: no difference between Eng+/-

and Eng+/+

Functional consequences

0

100

200

300

400

500

600

n=6 n=6

P=0.00450

05

1015202530354045

n=8 n=7

P=0.001

Vessels / mm2 Ejection fraction (%)

0

0.2

0.4

0.6

0.8

1

1.2

P=0.001

n=8 n=7

Cardiac index (l/min/kg)

Eng+/+

Eng+/-

• Neoangiogenesis defect in Eng+/- mice post-MI associated with impaired heart function

• Neoangiogenesis defect in Eng+/- mice post-MI associated with impaired heart function

• Partial rescue by injection of healthy MNCs

Functional consequences

0

100

200

300

400

500

600

n=6 n=6 n=7

P=0.003P=0.004

P=0.568

50

05

1015202530354045

n=8 n=7 n=7

P=0.004P=0.001

P=0.728

Vessels / mm2 Ejection fraction (%)

0

0.2

0.4

0.6

0.8

1

1.2

P=0.655P=0.001

P=0.132

n=8 n=7 n=7

Cardiac index (l/min/kg)

Eng+/+

Eng+/-

Eng+/-

+ MNCs

MNC defects

0

100

200

300

400

500

600

700P=0.013P=0.028

P=0.884

n=5 n=5 n=8

Vessels / mm2

0102030405060708090

100

n=9 n=10

P=0.003

UEA-1 / mm2

UEA-1PECAMHealthy

donor MNC

HHT1 patient MNC

PBS

• HHT1-MNCs fail to stimulate neoangiogenesis and to accumulate in the infarct region of Eng+/+ mice

Conclusion

• Defective vascular repair –mediated by MNCs- is a significant component of the etiology of HHT1

• This may explain disease heterogeneity, since exposure to vascular damage or inflammation varies between patients

• In general, MNC characteristics in any patient may affect their efficiency of vascular repair

Acknowledgements

• Hubrecht Laboratory– Christine Mummery– Franck Lebrin– Sander van den Driesche– Alie Feijen– Mariette Driessens

• Heart Lung Center Utrecht– Pieter Doevendans– Marie-José Goumans– Simone Post– Maurits Jansen– Cees van Echteld

• St Antonius Hospital Nieuwegein– Cees Westerman– Repke Snijder– Johannes Mager

• Leiden University Medical Center – Peter ten Dijke

• University of Newcastle– Helen Arthur