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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
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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
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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
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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
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MI in mice
• Myocardial infarction or
• Sham thoracotomy
• 4 week survival >60%
• Endoglin heterozygous mice (Eng+/-)• Wildtype littermates (Eng+/+)• Wildtype Balb/C
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MNC injection
• Venous blood from HHT1 patients or healthy volunteers
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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
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Analysis
• Endoglin expression infarcted vs. healthy heart (human and mouse): ISH and IHC
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Analysis
• Endoglin expression infarcted vs. healthy heart (human and mouse): ISH and IHC
• Homing human MNC to infarct : cryosections – UEA-1 lectin+FITC (4d)
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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)
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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)
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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)
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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
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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)
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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+/+
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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
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• 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
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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
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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
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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