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Angiogenesis

pp 1–15 | Cite as

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

  • Evgenii Boriushkin
  • Hui Zhang
  • Mitchell Becker
  • Janet Peachey
  • Mohammad A. Shatat
  • Ralf H. Adams
  • Anne Hamik
Original Paper

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.

Keywords

Angiogenesis Notch signaling Endothelium Kruppel-like factor 4 Transcriptional regulation 

Notes

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.

Author 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.

Supplementary material

10456_2018_9657_MOESM1_ESM.docx (12 kb)
Supplementary material 1 (DOCX 11 KB)
10456_2018_9657_MOESM2_ESM.pdf (1.2 mb)
Supplementary material 2 (PDF 1 MB)

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Copyright information

© This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2019

Authors and Affiliations

  1. 1.Department of MedicineStony Brook UniversityStony BrookUSA
  2. 2.Department of Physiology & Biophysics, School of MedicineCase Western Reserve UniversityClevelandUSA
  3. 3.Department of ChemistryUniversity of Michigan College of Literature, Science and ArtsAnn ArborUSA
  4. 4.Case Cardiovascular Research Institute, Case Western Reserve University, and Harrington Heart and Vascular InstituteUniversity Hospitals Cleveland Medical CenterClevelandUSA
  5. 5.Division of Pulmonary, Critical Care and Sleep MedicineCase Western Reserve UniversityClevelandUSA
  6. 6.Louis Stokes Cleveland VA Medical CenterClevelandUSA
  7. 7.Department of Tissue Morphogenesis, Faculty of Medicine, Max Planck Institute for Molecular BiomedicineUniversity of MünsterMünsterGermany
  8. 8.Institute for Molecular CardiologyStony Brook UniversityStony BrookUSA
  9. 9.Northport Veterans Affairs Medical CenterNorthportUSA
  10. 10.Department of MedicineStony Brook UniversityStony BrookUSA

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