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Endoglin prevents vascular malformation by regulating flow-induced cell migration and specification through VEGFR2 signalling


Loss-of-function (LOF) mutations in the endothelial cell (EC)-enriched gene endoglin (ENG) cause the human disease hereditary haemorrhagic telangiectasia-1, characterized by vascular malformations promoted by vascular endothelial growth factor A (VEGFA). How ENG deficiency alters EC behaviour to trigger these anomalies is not understood. Mosaic ENG deletion in the postnatal mouse rendered Eng LOF ECs insensitive to flow-mediated venous to arterial migration. Eng LOF ECs retained within arterioles acquired venous characteristics and secondary ENG-independent proliferation resulting in arteriovenous malformation (AVM). Analysis following simultaneous Eng LOF and overexpression (OE) revealed that ENG OE ECs dominate tip-cell positions and home preferentially to arteries. ENG knockdown altered VEGFA-mediated VEGFR2 kinetics and promoted AKT signalling. Blockage of PI(3)K/AKT partly normalized flow-directed migration of ENG LOF ECs in vitro and reduced the severity of AVM in vivo. This demonstrates the requirement of ENG in flow-mediated migration and modulation of VEGFR2 signalling in vascular patterning.

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Figure 1: Postnatal EC-specific Eng LOF causes local AVM and secondary sprouting.
Figure 2: Eng expression is suppressed in tip versus stalk cells, but promotes tip-cell potential.
Figure 3: Eng LOF reduces EC migration in sprouting angiogenesis.
Figure 4: Eng LOF mediates context-dependent effects on proliferation.
Figure 5: Loss or gain of ENG alters retinal EC distribution and Eng deletion initiates AVM in arterioles.
Figure 6: ENG regulates EC migration in response to shear stress and blood flow.
Figure 7: ENG affects VEGFR2 trafficking and downstream signalling.
Figure 8: Effects of VEGFR2 or PI(3)K inhibition on AVM properties and summary of concepts.

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We thank R. Adams (Max Planck Institute for Molecular Biomedicine, Germany) for providing the Cdh5(PAC)-CreERT2 mice, C. Vary (Michigan State University, USA) for the ENGiOE mice, G. Thurston (Regeneron, USA) for providing the EngLacZ mice, and B. Lavina and K. Gaengel (Uppsala University, Sweden) for sharing mice. We are grateful to Y. Xiong for help on the construction and package of lentivirus and to A. Keller, M. A. Mäe and K. Pietras for initial contributions. This study was supported by grants from William K. Bowes Jr Foundation (L.J.), the Swedish Research Council (L.J., C.B.), the Swedish Cancer Society (L.J., C.B.), the Cardiovascular Programme and the Strategic Research Programme in Neuroscience at Karolinska Institutet (L.J.), Jeanssons Stiftelser (L.J.), Magnus Bergvalls Stiftelse (L.J.), Knut and Alice Wallenbergs Stiftelse (C.B.), the European Research Council (C.B.), the Leducq Foundation (C.B.) and the British Heart Foundation (H.M.A.; RG/12/2/29416).

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L.J., Y.J. and L.M. designed the research; Y.J., L.M., C.B., H.M.A. and L.J. wrote the paper; Y.J., L.M., Y.W., M.B. and A.-C.D. performed the experiments and together with L.J. analysed the data. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Lars Jakobsson.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Confirmation of Eng deletion in the brain vasculature of EngiΔEC mice, and detection of hypoxia in retinas and vascular tufts formation in brain.

(a) Brain vasculature of a P7 EngiΔEC mouse induced for gene-deletion by tamoxifen at P1. The conditional expression of YFP (R26R locus) indicates Cre-mediated recombination (green) that negatively correlates with immunolabelling for ENG (red) within the complete vasculature (CD31, white). Most YFP positive cells in EngiΔEC mice have lost ENG. Images are representative of at least 5 brains analysed. Scale bars, 50 μm. (b) Immunostaining of CD31 (green) and pimonidazole adducts (hypoxia probe, red) in a P7 retina of Engflox/wt or EngiΔEC mouse. The dotted box indicates a region of hyper-sprouting co-localizing with increased hypoxia. Images are representative of 6 samples analysed. Scale bars, 400 μm. (c) A brain vascular glomeruloid tuft in an EngiΔEC mouse with mosaic recombination. CD31 (red), YFP (green). Images are representatives of at least 3 glomeruloid tufts analysed. Scale bars, 20 μm.

Supplementary Figure 2 Detection of Eng gene promoter activity by X-gal staining in neonatal or adult EnglacZ/wt mice.

(a) Sprouting brain vasculature of EnglacZ/wt mice at P7, stained for the endothelium (CD31, red; left) following X-gal reaction to detect Eng expression (black; right). Eng expression is lower in tip cells (arrow) than in adjacent stalk cells (arrow head). Images are representative of 3 brains analysed. Scale bars, 20 μm. (b) Retinas from EnglacZ/wt mice stained for CD31, (red) and submerged to an X-gal reaction (black). Dotted yellow line indicates the sprouting front of the superficial layer of the developing retinal vasculature. An artery and a vein are indicated with red and blue asterisks respectively. Images are representative of 4 retinas analysed. Scale bars, 100 μm. (c) Eng expression in arterioles in the adult retina of an EnglacZ/wt shown by X-gal reaction (black) following immunostaining for CD31 (green) and smooth muscle actin (ASMA, red). Images are representative of 4 retinas analysed. Scale bars, 50 μm.

Supplementary Figure 3 Confirmation of mosaic Eng overexpression in EngiΔEC mice and the specificity of Claudin5-GFP in ECs.

(a) Images from P7 retinas of EngiΔEC+iOE mice, showing complete recombination for Eng LOF (YFP, green) while only partial recombination for human ENG (red) transgene. CD31 staining (blue) visualized all vessels. Images are representative of at least 10 retinas analysed. Scale bars, 100 μm. (b) CD31 (red) staining in P7 Claudin5-GFP mouse brain shows complete overlap with endogenous GFP. Images are representative of 10 brain areas from 2 mice. Scale bar, 75 μm.

Supplementary Figure 4 Confirmation of the expression of H2B-mCherry in ECs in aortic explants upon induction of recombination.

Aortic explant from transgenic mice R26R-H2B-mCherry: Cdh5(PAC)-CreERT2. ECs are shown by immunostaining for CD31 (green) and ERG (EC specific nuclear protein, blue). EC specific expression of the conditional H2B-mCherry (red) upon tamoxifen treatment was shown by co-localisation with ERG. All cells have recombined due to tamoxifen induction. Images are representative of 8 aortic rings analysed. Scale bars, 50 μm.

Supplementary Figure 5 Rescuing AVM by introducing human ENG overexpression in EngiΔEC mice and the developing time course as well as the venous property of AVM.

(a) The development of AVMs in the P7 EngiΔEC mice (left) was rescued by the over-expression of human ENG in the EngiΔEC+iOE littermates (right). Images are representative of 2 mice for each genotype. Scale bars, 500 μm. (b) Progress of AVM development was shown by staining of retinal vasculature of EngiΔEC littermates at P6, P7, P8 and P10, induced for mosaic recombination at P4. Arrows indicate malformations. Images are representative of 2-8 retinas analysed at each time point. Scale bars, 200 μm. (c) Low Dll4 expression in AVM. Dll4 expression in an AVM in retina from P7 mouse is shown by immunostaining (red). Vessels are shown by the staining of CD31 (blue). Images are representative of 5 AVMs analysed. Scale bars, 100 μm.

Supplementary Figure 6 Partial altered EC-polarization in retinas of P7 EngiΔEC mice.

(ac) Polarization of the ECs was determined by the relative positioning of Golgi apparatus (GM130, green) to the nuclei. ECs with Cre-mediated recombination were shown by nuclear mCherry. All EC nuclei were stained for ERG (blue). Arrows indicate the Golgi apparatus of the ECs polarized against flow. Dashed line arrows indicate the Golgi apparatus of the ECs polarized with flow. Arrowheads indicate Golgi apparatus of the ECs not well polarized. (df) Quantifications of EC polarization in the arterial vessels in control mice (n = 13 vessels), non-AVM arterial vessels (n = 7 vessels, P = 0.049 (‘other’); P = 0.04 (‘against flow’), between Eng WT and Eng LOF, two-tailed unpaired t-test.) and AVMs in EngiΔEC mice (n = 8 vessels). Cells that polarize neither against nor with the flow direction are defined as ‘other’. Scale bars, 50 μm (whole pictures); 10 μm (boxed area).Values are mean ± s.e.m. NS, not significant.

Supplementary Figure 7 Vesicular co-localisation of VEGFR2 and ENG in the mouse brain vasculature and HDMECs.

(a) Co-staining for VEGFR2 (green) and ENG (red) of a brain-slice of a P7 WT mouse indicates partial co-localisation of VEGFR2 and ENG (arrowheads, 0.42 μm optical section). Arrows indicate VEGFR2 which are not co-localised with ENG. Images are representative of 5 fields analysed. Scale bars, 2 μm. (b) Staining for endosomal proteins EEA1, Rab5, Rab7 and a marker for endoplasmic reticulum (ER, Calnexin) together with VEGFR2 and ENG in VEGFA treated (15 min) HDMECs. VEGFR2 and ENG are frequently co-localised in EEA1, Rab5 or Rab7 positive endosomes but rarely in ER (boxed regions). Arrows indicate examples of VEGFR2 and ENG positive vesicles which are not co-localised with the endosomal markers above. Images are representative of 5 fields analysed. Scale bars, 10 μm (whole pictures); 1 μm (boxed area).

Supplementary Figure 8 Effects of VEGFR2 or PI3K inhibition on migration of ECs exposed to flow.

(a) Migration speed of control or ENG knockdown HDMECs under flow, untreated (data derive from sets in Fig. 6b), treated with SU5416 or Wortmannin was measured by MtrackJ in ImageJ. Control, shControl, n = 295 cells; shENG, n = 287 cells. SU5416, shControl, n = 103 cells; shENG, n = 108 cells. Wortmannin, shControl, n = 100 cells; shENG, n = 105 cells. shControl, P < 0.001 (SU5416); P = 0.006 (Wortmannin). shENG, P < 0.001 (both treatments). (b) The displacement of HDMECs migration towards direction of flow (Y axis) untreated (data derive from data sets in Fig. 6b), under the treatment of SU5416 or Wortmannin. Control, shControl, n = 295 cells; shENG, n = 287 cells. SU5416, shControl, n = 103 cells; shENG, n = 108 cells. Wortmannin, shControl, n = 100 cells; shENG, n = 105 cells, from one series of experiments. shControl, P = 0.01 (SU5416). shENG, P < 0.001 (both treatments). Values are population mean ± s.e.m. NS, not significant. P values in (a,b) indicate difference to the untreated control of the same genotype, two-tailed unpaired t-test.

Supplementary Figure 9 Unprocessed scans of western blots.

The whole membranes exposed for the detection of protein bands shown in Fig. 7 (a,c,e,h) are displayed in (a), (b), (c), (d), respectively.

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Supplementary Table 1

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3D-visualization of individual EC behaviour in the mouse aortic ring assay.

See Methods for the setup for imaging. Images were processed in Imaris for surface rendering of mCherry-labelled endothelial nuclei (green). Migratory routes of the ECs are shown with time-indicating tracks. Note the differences in the migratory speed and direction of individual ECs at different time. (AVI 14325 kb)

Migration of control HDMECs under flow.

See Methods for details on microscopy. Coloured dots and tracks are the migratory routes of all the trackable cells in a randomly chosen field. (AVI 8500 kb)

Migration of ENG knockdown HDMECs under flow.

See Methods for details on microscopy. Coloured dots and tracks are the migratory routes of all the trackable cells in a randomly chosen field. (AVI 9268 kb)

Analysis of EC migration in vessels of the wounded mouse cornea, by intravital imaging.

In vivo imaging of newly formed vessels in the mouse cornea 5 days after suturing. YFP expression (green) was induced in a subpopulation of ECs. Blood flow was visualized by transmitted light (100 ms/frame). Arrows indicate leukocytes rolling with the blood flow. (AVI 2616 kb)

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Jin, Y., Muhl, L., Burmakin, M. et al. Endoglin prevents vascular malformation by regulating flow-induced cell migration and specification through VEGFR2 signalling. Nat Cell Biol 19, 639–652 (2017).

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