The Krüppel-like factor 2 and Krüppel-like factor 4 genes interact to maintain endothelial integrity in mouse embryonic vasculogenesis

Abstract

BackgroundKrüppel-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.ResultsEndothelial 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.ConclusionsThis 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.

Epigenetic mechanisms underlying maternal diabetes-associated risk of congenital heart disease

“…In parallel to this, our findings from in vitro ATAC-seq revealed that Nos3 open chromatin regions (R1-R3) also contain binding sites for KLFs, SP1, and the ETS-family of TFs, which can regulate Nos3's expression through another feedback mechanism under HG stress (Supplemental Tables 5-7). This observation is supplemented by the fact that Klf2 and Klf4 ablation in embryos resulted in EC disruption by reducing Nos3 expression (60). Further characterization of these TFs on Nos3 enhancer regulation upon HG exposure is needed.…”

KLF2 and KLF4 control endothelial identity and vascular integrity

“…We also note that our findings are consistent with but add significantly to previous findings. Specifically, Chiplunkar et al (13) reported the role of both factors in regulating vascular integrity using systemic knockout embryos of both Klf2 and Klf4 genes. However, their study used a global deletion of Klf2 and Klf4 genes in the embryos and focused on early development.…”

Krüppel-like factors in mammalian stem cells and development

“…It was further shown that the lack of Klf4 in these mice decreased the expression of many cardiac genes, including myosin light polypeptide 1 (Myl1), cardiac/slow skeletal troponin C (Tnnc1), myosin binding protein C (Mybpc2), natriuretic peptide precursor type B (Nppb) and Gata4 (Yoshida et al, 2010). An independent study showed that Klf2/Klf4 double knockout mice die earlier than the single knockouts, possibly due to gross hemorrhaging of multiple vessels (Chiplunkar et al, 2013a). Thus, KLF4 might play a role in vasculogenesis and the maintenance of embryonic vascular integrity by interacting with KLF2.…”