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

Chiplunkar AR, Curtis BC, Eades GL, Kane MS, Fox SJ, Haar JL, Lloyd JA - BMC Dev. Biol. (2013)

Bottom Line: 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.Down-regulation of these genes in KLF2-/-KLF4-/- embryos may result in the observed loss of vascular integrity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, Virginia 23298-0035, USA. jlloyd@vcu.edu.

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.

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KLF2-/-KLF4-/- primary head vein lacks continuous endothelial layer at E9.5. Light micrographs of 6 μm sections were taken at 200X magnification. A) WT and B) KLF2-/- (n = 4) shows normal looking primary head vein; C) Two of the four KLF2-/-KLF4+/- embryos have gaps in the endothelial layer of the primary head vein (n = 4); D) KLF2-/-KLF4-/- primary head vein lacks a continuous endothelial layer (n = 3). Red arrows indicate apparent gaps in the endothelial layer. Black arrows point at the lumen of the Primary Head Vein (PHV).
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Figure 2: KLF2-/-KLF4-/- primary head vein lacks continuous endothelial layer at E9.5. Light micrographs of 6 μm sections were taken at 200X magnification. A) WT and B) KLF2-/- (n = 4) shows normal looking primary head vein; C) Two of the four KLF2-/-KLF4+/- embryos have gaps in the endothelial layer of the primary head vein (n = 4); D) KLF2-/-KLF4-/- primary head vein lacks a continuous endothelial layer (n = 3). Red arrows indicate apparent gaps in the endothelial layer. Black arrows point at the lumen of the Primary Head Vein (PHV).

Mentions: Tissue-sections of the PHV at the level of the optic vesicle reveal a continuous endothelium in E9.5 WT (Figure 2A) and KLF2-/- (Figure 2B) embryos. In KLF2-/-KLF4-/- embryos, there are apparent gaps between adjoining endothelial cells (Figure 2D). Endothelial disruption of the PHV, which was not evident at the gross level, is also seen at the microscopic level in two of the four KLF2-/-KLF4+/- embryos that were examined (Figure 2C). This phenotype is variable, and the other two E9.5 KLF2-/-KLF4+/- embryos appeared like wild-type, having no PHV phenotype. The presence of apparent gaps in the endothelial layer suggests a lack of vascular integrity. No abnormal phenotype was seen in KLF2+/-KLF4-/- embryos (data not shown), suggesting that complete KLF2 ablation is required for this abnormal vascular phenotype. The fact that the KLF2-/-KLF4-/- and KLF2-/-KLF4+/- phenotypes are more severe than KLF2-/- indicates that KLF4 plays a larger role in vascular development than previously recognized and is complemented by KLF2. Serial tissue sections of entire E9.5 embryos were examined for this study, and the abnormal vascular phenotype in KLF2-/-KLF4-/- and some KLF2-/-KLF4+/- embryos is observed only in the primary head vein. The primary head vein is one of the major blood vessels at this stage of development. Although the PHV is the only vessel to exhibit hemorrhaging at E9.5, based on the gross hemorrhaging observed by E10.5, other vessels in KLF2-/-KLF4-/- embryos lack integrity by this later time point.


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

Chiplunkar AR, Curtis BC, Eades GL, Kane MS, Fox SJ, Haar JL, Lloyd JA - BMC Dev. Biol. (2013)

KLF2-/-KLF4-/- primary head vein lacks continuous endothelial layer at E9.5. Light micrographs of 6 μm sections were taken at 200X magnification. A) WT and B) KLF2-/- (n = 4) shows normal looking primary head vein; C) Two of the four KLF2-/-KLF4+/- embryos have gaps in the endothelial layer of the primary head vein (n = 4); D) KLF2-/-KLF4-/- primary head vein lacks a continuous endothelial layer (n = 3). Red arrows indicate apparent gaps in the endothelial layer. Black arrows point at the lumen of the Primary Head Vein (PHV).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222490&req=5

Figure 2: KLF2-/-KLF4-/- primary head vein lacks continuous endothelial layer at E9.5. Light micrographs of 6 μm sections were taken at 200X magnification. A) WT and B) KLF2-/- (n = 4) shows normal looking primary head vein; C) Two of the four KLF2-/-KLF4+/- embryos have gaps in the endothelial layer of the primary head vein (n = 4); D) KLF2-/-KLF4-/- primary head vein lacks a continuous endothelial layer (n = 3). Red arrows indicate apparent gaps in the endothelial layer. Black arrows point at the lumen of the Primary Head Vein (PHV).
Mentions: Tissue-sections of the PHV at the level of the optic vesicle reveal a continuous endothelium in E9.5 WT (Figure 2A) and KLF2-/- (Figure 2B) embryos. In KLF2-/-KLF4-/- embryos, there are apparent gaps between adjoining endothelial cells (Figure 2D). Endothelial disruption of the PHV, which was not evident at the gross level, is also seen at the microscopic level in two of the four KLF2-/-KLF4+/- embryos that were examined (Figure 2C). This phenotype is variable, and the other two E9.5 KLF2-/-KLF4+/- embryos appeared like wild-type, having no PHV phenotype. The presence of apparent gaps in the endothelial layer suggests a lack of vascular integrity. No abnormal phenotype was seen in KLF2+/-KLF4-/- embryos (data not shown), suggesting that complete KLF2 ablation is required for this abnormal vascular phenotype. The fact that the KLF2-/-KLF4-/- and KLF2-/-KLF4+/- phenotypes are more severe than KLF2-/- indicates that KLF4 plays a larger role in vascular development than previously recognized and is complemented by KLF2. Serial tissue sections of entire E9.5 embryos were examined for this study, and the abnormal vascular phenotype in KLF2-/-KLF4-/- and some KLF2-/-KLF4+/- embryos is observed only in the primary head vein. The primary head vein is one of the major blood vessels at this stage of development. Although the PHV is the only vessel to exhibit hemorrhaging at E9.5, based on the gross hemorrhaging observed by E10.5, other vessels in KLF2-/-KLF4-/- embryos lack integrity by this later time point.

Bottom Line: 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.Down-regulation of these genes in KLF2-/-KLF4-/- embryos may result in the observed loss of vascular integrity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, Virginia 23298-0035, USA. jlloyd@vcu.edu.

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.

Show MeSH
Related in: MedlinePlus