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Kruppel-like factor 4 regulates developmental angiogenesis through disruption of the RBP-J–NICD–MAML complex in intron 3 of Dll4

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Abstract

Angiogenesis is a multistep process that requires highly regulated endothelial cell (EC) behavior. The transcription factor Krüppel-like factor 4 (KLF4) is a critical regulator of several basic EC functions; we have recently shown that KLF4 disturbs pathological (tumor) angiogenesis by mediating the expression of members of VEGF and Notch signaling pathways. Notch signaling is central to orchestration of sprouting angiogenesis but little is known about the upstream regulation of Notch itself. To determine the role of KLF4 in normal (developmental) angiogenesis, we used a mouse retinal angiogenesis model. We found that endothelial-specific overexpression of KLF4 in transgenic mice (EC-K4 Tg) leads to increased vessel density, branching and number of tip cell filopodia as assessed on postnatal day 6 (P6). The hypertrophic vasculature seen with sustained KLF4 overexpression is not stable and undergoes prominent remodeling during P7–P12 resulting in a normal appearing retinal vasculature in adult EC-K4 Tg mice. We find that KLF4 inhibits Delta-like 4 (DLL4) expression in the angiogenic front during retinal vascular development. Furthermore, in an oxygen-induced retinopathy model, overexpression of KLF4 results in decreased vaso-obliteration and neovascular tuft formation that is similar to genetic or pharmacologic DLL4 inhibition. Mechanistically, we show that KLF4 disables the activity of the essential Notch transcriptional activator RBP-J by interfering with binding of co-activators NICD and MAML at intron 3 of the Notch ligand DLL4. In summary, our experimental results demonstrate a regulatory role of KLF4 in developmental angiogenesis through regulation of DLL4 transcription.

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Acknowledgements

We are grateful for the support and encouragement provided by Dr. Mukesh K. Jain. We thank Dr. Christina Antonopoulos-Buzzy (Case Western Reserve University School of Medicine) for kindly providing technical support, discussion, and review of the manuscript. We thank Kevin P. Montgomery for assistance with figures. This work is supported by NIH R01HL113570.

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Authors and Affiliations

  1. Department of Medicine, Stony Brook University, Stony Brook, NY, 11794, USA

    Evgenii Boriushkin & Anne Hamik

  2. Department of Physiology & Biophysics, School of Medicine, Case Western Reserve University, Cleveland, OH, 44106, USA

    Hui Zhang

  3. Department of Chemistry, University of Michigan College of Literature, Science and Arts, Ann Arbor, MI, 48104, USA

    Mitchell Becker

  4. Case Cardiovascular Research Institute, Case Western Reserve University, and Harrington Heart and Vascular Institute, University Hospitals Cleveland Medical Center, Cleveland, OH, 44106, USA

    Janet Peachey

  5. Division of Pulmonary, Critical Care and Sleep Medicine, Case Western Reserve University, Cleveland, OH, 44106, USA

    Mohammad A. Shatat

  6. Louis Stokes Cleveland VA Medical Center, Cleveland, OH, 44106, USA

    Mohammad A. Shatat

  7. Department of Tissue Morphogenesis, Faculty of Medicine, Max Planck Institute for Molecular Biomedicine, University of Münster, 48149, Münster, Germany

    Ralf H. Adams

  8. Institute for Molecular Cardiology, Stony Brook University, Stony Brook, NY, 11794, USA

    Anne Hamik

  9. Northport Veterans Affairs Medical Center, Northport, NY, 11768, USA

    Anne Hamik

  10. Department of Medicine, Stony Brook University, 100 Nicolls Rd, HSC T-15, Room 048, Stony Brook, NY, 11794, USA

    Anne Hamik

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  1. Evgenii Boriushkin
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Contributions

EB designed, performed, and analyzed experiments, and drafted the manuscript. HZ, JP, and MAS provided input into experimental results and reviewed the manuscript. RHA provided crucial reagents and reviewed the manuscript. AH conceived and coordinated the studies, designed, and analyzed the experiments, and revised the manuscript. All authors approved the final version of the manuscript.

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Correspondence to Anne Hamik.

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Boriushkin, E., Zhang, H., Becker, M. et al. Kruppel-like factor 4 regulates developmental angiogenesis through disruption of the RBP-J–NICD–MAML complex in intron 3 of Dll4. Angiogenesis 22, 295–309 (2019). https://doi.org/10.1007/s10456-018-9657-y

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  • DOI: https://doi.org/10.1007/s10456-018-9657-y

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