title: The Krüppel-like factor 2 and Krüppel-like factor 4 genes interact to maintain endothelial integrity in mouse embryonic vasculogenesis.
abstract: BACKGROUND
  Krüppel-like Factor 2 (KLF2) plays an important role in vessel maturation during embryonic development. In adult mice, KLF2 regulates expression of the tight junction protein occludin, which may allow KLF2 to maintain vascular integrity. Adult tamoxifen-inducible Krüppel-like Factor 4 (KLF4) knockout mice have thickened arterial intima following vascular injury. The role of KLF4, and the possible overlapping functions of KLF2 and KLF4, in the developing vasculature are not well-studied.
  
  
  RESULTS
  Endothelial breaks are observed in a major vessel, the primary head vein (PHV), in KLF2-/-KLF4-/- embryos at E9.5. KLF2-/-KLF4-/- embryos die by E10.5, which is earlier than either single knockout. Gross hemorrhaging of multiple vessels may be the cause of death. E9.5 KLF2-/-KLF4+/- embryos do not exhibit gross hemorrhaging, but cross-sections display disruptions of the endothelial cell layer of the PHV, and these embryos generally also die by E10.5. Electron micrographs confirm that there are gaps in the PHV endothelial layer in E9.5 KLF2-/-KLF4-/- embryos, and show that the endothelial cells are abnormally bulbous compared to KLF2-/- and wild-type (WT). The amount of endothelial Nitric Oxide Synthase (eNOS) mRNA, which encodes an endothelial regulator, is reduced by 10-fold in E9.5 KLF2-/-KLF4-/- compared to KLF2-/- and WT embryos. VEGFR2, an eNOS inducer, and occludin, a tight junction protein, gene expression are also reduced in E9.5 KLF2-/-KLF4-/- compared to KLF2-/- and WT embryos.
  
  
  CONCLUSIONS
  This study begins to define the roles of KLF2 and KLF4 in the embryonic development of blood vessels. It indicates that the two genes interact to maintain an intact endothelial layer. KLF2 and KLF4 positively regulate the eNOS, VEGFR2 and occludin genes. Down-regulation of these genes in KLF2-/-KLF4-/- embryos may result in the observed loss of vascular integrity.
authors.affiliation: Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, Virginia 23298-0035, USA. jlloyd@vcu.edu.
authors.forename: Aditi R
authors.initials: AR
authors.lastname: Chiplunkar
authors.forename: Benjamin C
authors.initials: BC
authors.lastname: Curtis
authors.forename: Gabriel L
authors.initials: GL
authors.lastname: Eades
authors.forename: Megan S
authors.initials: MS
authors.lastname: Kane
authors.forename: Sean J
authors.initials: SJ
authors.lastname: Fox
authors.forename: Jack L
authors.initials: JL
authors.lastname: Haar
authors.forename: Joyce A
authors.initials: JA
authors.lastname: Lloyd
mesh_terms: D000818:Animals; D001808:Blood Vessels / Q000196:embryology*; D004622:Embryo, Mammalian; D047108:Embryonic Development; D004730:Endothelium, Vascular / Q000196:embryology* / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D020300:Intracranial Hemorrhages / Q000196:embryology / Q000378:metabolism; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D008854:Microscopy, Electron; D009024:Morphogenesis; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism; D062793:Occludin / Q000235:genetics; D051497:Receptor, Fibroblast Growth Factor, Type 2 / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013629:Tamoxifen / Q000494:pharmacology
chemical_list: C000715467:KLF4 protein, human; C498314:Klf2 protein, mouse; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D062793:Occludin; C568986:Ocln protein, mouse; D013629:Tamoxifen; D019001:Nitric Oxide Synthase; C496354:Fgfr2 protein, mouse; D051497:Receptor, Fibroblast Growth Factor, Type 2