Endothelial EphB4 maintains vascular integrity and transport function in adult heart
- Max Planck Institute for Molecular Biomedicine, Germany
- University Hospital Münster, Germany
- University of Münster, Germany
- AstraZeneca, Sweden
Figures
Figure 1 with 7 supplements
Heart defects in adult Ephb4 mutants.
(A) Freshly dissected Ephb4∆EC and littermate control hearts at 12 weeks of age. Heart weight and heart weight/tibia length index (HW/TL) ratio are increased in Ephb4∆EC mutants, whereas body weight …
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Figure 1—source data 1
Source data for Figure 1A,B,C,D.
- https://cdn.elifesciences.org/articles/45863/elife-45863-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Inactivation of Ephb4 in adult ECs.
(A–D) Immunohistochemistry on cross sections of hearts. Panels show the outer part of the wall of the left ventricle. (A) EphB4 is expressed in veins, venules (white arrowheads) and small …
Figure 1—video 1
Left ventricle long axis view.
Figure 1—video 2
Left ventricle long axis view.
Figure 1—video 3
Four chamber view.
Figure 1—video 4
Short axis view.
Figure 1—video 5
Four chamber view.
Figure 1—video 6
CMRI 12 week-old Ephb4∆EC mutant.
Short axis view.
Figure 2 with 3 supplements
Ephb4 inactivation compromises cardiac vascular integrity.
(A–C) Immunostaining of cross sections through 12 week-old control and Ephb4∆EC hearts. Confocal images show the outer (A, C) and inner part (B) of the left ventricle. (A) Vascular density, measured …
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Figure 2—source data 1
Source data for Figure 2A,B.
- https://cdn.elifesciences.org/articles/45863/elife-45863-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Analysis of cell death and hypoxia.
(A) TUNEL assay in Ephb4∆EC hearts. There is no detectable cell death in mutant ECs of the ventricular wall. (B) Hypoxia assay using Pimonidazole shows that Ephb4∆EC hearts are not hypoxic. Hearts …
Figure 2—figure supplement 2
Ephb4 inactivation does not induce fibrosis in heart.
(A) Immunohistochemistry on for Collagen I (ColI, green) and Collagen IV (ColIV, green) in transverse sections of control and Ephb4∆EC heart showing both ventricles. Arrowheads mark normal signal. …
Figure 2—figure supplement 3
Ultrastructural analysis of Ephb4 mutant hearts.
(A–C) Electron micrographs show details of the ventricular wall of the left ventricle. (A) Mutant endothelial cells are thickened and present irregular lining (arrowheads). (B) Accumulation of …
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Figure 2—figure supplement 3—source data 1
Source data for Figure 2—figure supplement 3C.
- https://cdn.elifesciences.org/articles/45863/elife-45863-fig2-figsupp3-data1-v1.xlsx
Figure 3 with 2 supplements
Heart phenotype after Efnb2 inactivation in the vascular endothelium.
(A) Efnb2∆EC and littermate control hearts at 12 weeks of age. Heart weight and heart weight/tibia length index (HW/TL) remain unaffected in Efnb2∆EC mutants. N = 6 for control and N = 5 for Efnb2∆EC…
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Figure 3—source data 1
Source data for Figure 3A,B,C,D.
- https://cdn.elifesciences.org/articles/45863/elife-45863-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Cardiomyocyte hypertrophy at 2 weeks after Ephb4 inactivation.
(A) Control and Ephb4∆EC hearts at 10 weeks of age. Heart weight and heart weight/tibia length index (HW/TL) are unchanged in mutants. (B–D) Immunohistochemistry on cross sections of 12 week-old …
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Figure 3—figure supplement 1—source data 1
Source data for Figure 3—figure supplement 1A,B,C,D.
- https://cdn.elifesciences.org/articles/45863/elife-45863-fig3-figsupp1-data1-v1.xlsx
Figure 3—figure supplement 2
Microhemorrhages in Ephb4 and Efnb2 mutant hearts.
(A, B) Immunostaining on cross sections of the outer wall of the left ventricle. (A) Presence of microhemorrhages in the myocardium of Ephb4∆EC mice at 10 weeks. (B) Presence of Ter119+ red blood …
Figure 4 with 2 supplements
EphB4 inactivation does not affect the skeletal muscle.
(A–D) Immunohistochemistry on longitudinal sections of control and Ephb4∆EC gastrocnemius at 12 weeks of age. (A) EphB4 and ephrin-B2 (GFP in Efnb2::GFP reporter line) are expressed in capillaries …
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Figure 4—source data 1
Source data for Figure 4B,C,D.
- https://cdn.elifesciences.org/articles/45863/elife-45863-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Liver and kidney vasculature after Ephb4 inactivation.
(A, B) Immunostaining on sections (150 µm thick) of the right kidney of 12 week-old Ephb4∆EC mutants and control littermates. No significant changes in kidney vasculature are visible after staining …
Figure 4—figure supplement 2
Lymphatic inactivation of Ephb4 in adults does not produce cardiac defects.
(A) Immunohistochemistry of lymphatic vessel in the intestinal mesentery. EphB4 immunosignal is lost in lymphatic vessels (arrowheads) but maintained in Ephb4∆LEC veins (arrows). (B) Control and …
Figure 5 with 1 supplement
RNA-seq analysis of total heart suggests a metabolic shift in Ephb4 mutants.
(A–F) Global gene expression analysis of Ephb4∆EC ventricles by RNA sequencing at 12 weeks. (A) MA plot representing the 529 differentially expressed genes (p<0.05) including 268 upregulated genes …
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Figure 5—source data 1
RNA-seq analysis of Ephb4∆EC and control mouse heart ventricles.
The table indicates all the transcripts significantly upregulated and downregulated in Ephb4 mutant hearts.
- https://cdn.elifesciences.org/articles/45863/elife-45863-fig5-data1-v1.xlsx
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Figure 5—source data 2
Global proteome analysis of Ephb4∆EC and control mouse heart ventricles.
The table indicates all the proteins significantly upregulated and downregulated in Ephb4 mutant hearts.
- https://cdn.elifesciences.org/articles/45863/elife-45863-fig5-data2-v1.xlsx
Figure 5—figure supplement 1
Genome and Proteome analysis of adult Ephb4 mutant ventricles.
(A–C) Global gene expression analysis of 12 week-old Ephb4∆EC ventricles by RNA sequencing. (A) Sample distance analysis of RNA-seq samples. N = 3 for controls and N = 3 for Ephb4∆EC. (B) TPM …
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Figure 5—figure supplement 1—source data 1
Source data for Figure 5—figure supplement 1B,C.
- https://cdn.elifesciences.org/articles/45863/elife-45863-fig5-figsupp1-data1-v1.xlsx
Figure 6
EphB4-induced signaling in cultured ECs.
(A) Western blot analysis of lysates from cultured HUVECs stimulated with control human IgG/Fc (Ctrl Fc), human ephrin-B2/Fc (B2/Fc) or mouse EphB4/Fc (B4/Fc) (4 µg/ml, preclustered with 10µg/ml …
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Figure 6—source data 1
Source data for Figure 6B.
- https://cdn.elifesciences.org/articles/45863/elife-45863-fig6-data1-v1.xlsx
Figure 7 with 1 supplement
Alterations after knockdown of EPHB4 expression in ECs.
(A) Western blot analysis of HUVECs transfected with siControl, siEPHB4 or siCAV1, as indicated. Knockdown cells showed a reduction of CAV1 and pCAV1 protein levels. N = 3 for all treatments. (B) …
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Figure 7—source data 1
Source data for Figure 7C.
- https://cdn.elifesciences.org/articles/45863/elife-45863-fig7-data1-v1.xlsx
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Figure 7—source data 2
Source data for Figure 7E.
- https://cdn.elifesciences.org/articles/45863/elife-45863-fig7-data2-v1.xlsx
Figure 7—figure supplement 1
CD36 localization in EPHB4 and CAV1 knockdown HUVECs.
(A) Confocal images of siRNA-transfected HUVECs stained with CD36 (green), Golgi (GOLPH4, red), VE-Cadherin (CDH5, white) and nuclei (DAPI; blue). (B) Quantitation of CD36 signals localized to Golgi …
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Figure 7—figure supplement 1—source data 1
Source data for Figure 7—figure supplement 1B.
- https://cdn.elifesciences.org/articles/45863/elife-45863-fig7-figsupp1-data1-v1.xlsx
Figure 8 with 1 supplement
EphB4 activation induces Src-dependent phosphorylation via Src.
(A) Western blot analysis of CAV1 and pCAV1 levels in HUVECs stimulated with control human IgG/Fc (Ctrl Fc), ephrin-B2/Fc (B2/Fc) or EphB4/Fc (B4/Fc) (4 µg/ml, preclustered with 10µg/ml goat …
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Figure 8—source data 1
Source data for Figure 8B,F.
- https://cdn.elifesciences.org/articles/45863/elife-45863-fig8-data1-v1.xlsx
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Figure 8—source data 2
Source data for Figure 8D.
- https://cdn.elifesciences.org/articles/45863/elife-45863-fig8-data2-v1.xlsx
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Figure 8—source data 3
Source data for Figure 8E.
- https://cdn.elifesciences.org/articles/45863/elife-45863-fig8-data3-v1.xlsx
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Figure 8—source data 4
Source data for Figure 8G.
- https://cdn.elifesciences.org/articles/45863/elife-45863-fig8-data4-v1.xlsx
Figure 8—figure supplement 1
Effect of Akt, ERK and Src inhibition on CAV1 and CD36 localization in stimulated HUVECs.
(A, B) Cell area quantitation of HUVECs transfected with siControl, siEPHB4 or siCAV1 (A) and HUVECs stimulated with control human IgG/Fc (Ctrl Fc), human ephrin-B2/Fc (B2/Fc) or mouse EphB4/Fc …
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Figure 8—figure supplement 1—source data 1
Source data for Figure 8—figure supplement 1A,B.
- https://cdn.elifesciences.org/articles/45863/elife-45863-fig8-figsupp1-data1-v1.xlsx
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Figure 8—figure supplement 1—source data 2
Source data for Figure 8—figure supplement 1C.
- https://cdn.elifesciences.org/articles/45863/elife-45863-fig8-figsupp1-data2-v1.xlsx
Figure 9 with 2 supplements
EphB4 is required for caveolar transport.
(A) Confocal images of HUVECs transfected siControl, siEPHB4 and siCAV1. Cells were fixed 30 min after exposure to BSA-555 (red) and immunostained with anti-CDH5 (white) antibody and DAPI (nuclei, …
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Figure 9—source data 1
Source data for Figure 9C.
- https://cdn.elifesciences.org/articles/45863/elife-45863-fig9-data1-v1.xlsx
Figure 9—figure supplement 1
BSA, transferrin and BODIPY uptake in HUVECs.
(A, B) Flow cytometric analysis of Alexa Fluor 555 dye-conjugated bovine serum albumin (BSA-555) uptake by HUVECs transfected with siControl and siEPHB4 (A). N = 5 for both conditions. Data …
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Figure 9—figure supplement 1—source data 1
Source data for Figure 9—figure supplement 1A,C,F,G.
- https://cdn.elifesciences.org/articles/45863/elife-45863-fig9-figsupp1-data1-v1.xlsx
Figure 9—figure supplement 2
Metabolic features of Ephb4∆EC mice.
(A) Analysis of plasma from Ephb4∆EC mutants and control mice. Glucose concentration is reduced in mutants, whereas the concentrations of Triglycerides and Free fatty acids are unaltered. N = 4 for …
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Figure 9—figure supplement 2—source data 1
Source data for Figure 9—figure supplement 2A.
- https://cdn.elifesciences.org/articles/45863/elife-45863-fig9-figsupp2-data1-v1.xlsx
Figure 10
EphB4 is required for endothelial integrity.
(A) Immunohistochemistry of cultured HUVECs. Relative colocalization of Vinculin and CDH5 is reduced in the cell contact area (arrowheads) of siEPHB4 cells. (B) Mechanical stress assay on cultured …
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Figure 10—source data 1
Source data for Figure 10C,D.
- https://cdn.elifesciences.org/articles/45863/elife-45863-fig10-data1-v1.xlsx
Additional files
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Supplementary file 1
Key Resources Table.
List of all the materials and resources used.
- https://cdn.elifesciences.org/articles/45863/elife-45863-supp1-v1.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/45863/elife-45863-transrepform-v1.docx
