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BMP9 induces EphrinB2 expression in endothelial cells through an Alk1-BMPRII/ActRII-ID1/ID3-dependent pathway: implications for hereditary hemorrhagic telangiectasia type II

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Abstract

ALK1 (ACVRL1) is a member of the TGFβ receptor family and is expressed predominantly by arterial endothelial cells (EC). Mutations in ACVRL1 are responsible for hereditary hemorrhagic telangiectasia type 2 (HHT2), a disease manifesting as fragile vessels, capillary overgrowth, and numerous arterio-venous malformations. Arterial EC also express EphrinB2, which has multiple roles in vascular development and angiogenesis and is known to be reduced in ACVRL1 knockout mice. Using an in vitro angiogenesis model we find that the Alk1 ligand BMP9 induces EphrinB2 in EC, and this is entirely dependent on expression of Alk1 and at least one of the co-receptors BMPRII or ActRII. BMP9 induces both ID1 and ID3, and both are necessary for full induction of EphrinB2. Loss of Alk1 or EphrinB2 results in increased arterial-venous anastomosis, while loss of Alk1 but not EphrinB2 results in increased VEGFR2 expression and enhanced capillary sprouting. Conversely, BMP9 blocks EC sprouting and this is dependent on Alk1, BMPRII/ActRII and ID1/ID3. Finally, notch signaling overcomes the loss of Alk1—restoring EphrinB2 expression in EC, and curbing excess sprouting. Thus, in an in vitro model of HHT2, loss of Alk1 blocks BMP9 signaling, resulting in reduced EphrinB2 expression, enhanced VEGFR2 expression, and misregulated EC sprouting and anastomosis.

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Acknowledgments

This work was supported by a grant from the HHT Foundation International (CCWH) and an NIH R01 Award (HL60067–CCWH), and an NIH RC1 Award (ES018361–SCG). We thank Dr. Paul Oh for the kind gift of Alk1 plasmids. We thank Linda Him and Matt Peacock for outstanding help with tissue culture.

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Authors and Affiliations

  1. Department of Molecular Biology and Biochemistry, University of California Irvine, Irvine, CA, 92697, USA

    Jai-Hyun Kim, Matthew R. Peacock & Christopher C. W. Hughes

  2. Department of Biomedical Engineering, University of California Irvine, Irvine, CA, 92697, USA

    Steven C. George & Christopher C. W. Hughes

  3. Edwards Lifesciences Center for Advanced Cardiovascular Technology, University of California Irvine, Irvine, CA, 92697, USA

    Steven C. George & Christopher C. W. Hughes

  4. Department of Chemical Engineering and Materials Science, University of California Irvine, Irvine, CA, 92697, USA

    Steven C. George

  5. Department of Medicine, University of California Irvine, Irvine, CA, 92697, USA

    Steven C. George

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  1. Jai-Hyun Kim
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Correspondence to Christopher C. W. Hughes.

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10456_2012_9277_MOESM1_ESM.tiff

Fig. S1 ECFC-EC have a phenotype between HUAEC and HUVEC HUAEC, ECFC-EC and HUVEC were grown under identical conditions and then harvested for analysis of artery (EphrinB2) and vein (EphB4, COUP-TFII) markers by qRT-PCR (TIFF 2703 kb)

10456_2012_9277_MOESM2_ESM.tiff

Fig. S2 Alk1 and notch independently regulate angiogenic sprouting A. HUAEC were transfected with expression plasmids for CA-Alk1 or control, established in a sprouting assay and treated with vehicle or 3 μM DAPT. Sprouting was assessed after 5 days by counting tip cells. B. HUAEC were transfected with siRNA to Alk1 or control, and a day later transfected with an expression plasmid encoding dll4, or with a control plasmid. Cells were then seeded in a sprouting assay and sprouts/bead assessed at day 5. (TIFF 2703 kb)

10456_2012_9277_MOESM3_ESM.tiff

Fig. S3 BMP9 suppresses sprouting induced by a loss of notch signaling A. HUAEC were established in a sprouting assay and treated with DAPT and/or BMP9 as indicated. Sprouting was assessed after 5 days by counting tip cells (TIFF 2703 kb)

10456_2012_9277_MOESM4_ESM.tiff

Fig. S4 Multiple BMP response elements in the EphrinB2 promoter Approximately 1 kb of the human EphrinB2 promoter (-900 to +100 bp) was analyzed. The RBPj site is shown in purple, the nine BMP-response elements in green, and the transcription start site is indicated in yellow (TIFF 2703 kb)

10456_2012_9277_MOESM5_ESM.tiff

Fig. S5 At least one conserved BMP response element in the EphrinB2 promoter in multiple species Overlapping regions of the EphrinB2 promoter were aligned from multiple species. At least one site was well conserved between them all – 6/6 residues conserved between human, chimpanzee, chicken and zebrafish, and 5/6 conserved with mouse (TIFF 2703 kb)

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Kim, JH., Peacock, M.R., George, S.C. et al. BMP9 induces EphrinB2 expression in endothelial cells through an Alk1-BMPRII/ActRII-ID1/ID3-dependent pathway: implications for hereditary hemorrhagic telangiectasia type II. Angiogenesis 15, 497–509 (2012). https://doi.org/10.1007/s10456-012-9277-x

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