Korean Circ J.  2012 Apr;42(4):239-245. 10.4070/kcj.2012.42.4.239.

Kruppel-Like Factor 2 Suppression by High Glucose as a Possible Mechanism of Diabetic Vasculopathy

• Lee HY
• Youn SW
• Oh BH
• Kim HS

• KMID: 2225016
• DOI: http://doi.org/10.4070/kcj.2012.42.4.239

Abstract

BACKGROUND AND OBJECTIVES
Endothelial dysfunction is widely observed in diabetes mellitus, resulting in diabetic vascular complications. Kruppel-like factor 2 (KLF2) is implicated as being a key molecule that maintains endothelial function. We evaluated the expression of KLF2 in endothelial cells cultured in high glucose and investigated its functional implication in a diabetic animal model.
SUBJECTS AND METHODS
Human umbilical vein endothelial cells (HUVECs) were cultured in physiologically high glucose (35 mM) condition. The Otsuka Long Evans Tokushima Fatty (OLETF) strain of rat was used as an excellent model of obese type II diabetes, and their lean littermates are Long Evans Tokushima Otsuka (LETO) rats.
RESULTS
In HUVECs cultured in physiologically high glucose condition, FOXO1 was activated whereas KLF2 and endothelial nitric oxide synthase (eNOS) expression was near completely abolished, which was completely reversed by FOXO1 small interfering ribonucleic acid. In the vessels harvested from the OLETF rats, the animal model of type II diabetes, KLF2 and eNOS expression were found depleted. When vascular remodeling was induced in the left common carotid artery by reduction of blood flow with partial ligation of the distal branches, greater neointimal hypertrophy was observed in OLETF rats compared with the control LETO rats.
CONCLUSION
KLF2 suppression in endothelial cells by high glucose is a possible mechanism of diabetic endothelial dysfunction. The strategy of replenishing KLF2 may be effective for preventing diabetic vascular dysfunction.

Keyword

Diabetes mellitus; Endothelial cells; Kruppel-like factor 2

MeSH Terms

Animals
Carotid Artery, Common
Diabetes Mellitus
Diabetic Angiopathies
Endothelial Cells
Glucose
Human Umbilical Vein Endothelial Cells
Hypertrophy
Ligation
Models, Animal
Nitric Oxide Synthase Type III
Rats
Rats, Inbred OLETF
RNA
Sprains and Strains
Glucose
Nitric Oxide Synthase Type III
RNA

Figure

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  Fig. 1 Morphologic changes of HUVECs 72 hours after culture under high glucose conditions. HUVECs were cultured in high glucose with the indicated concentration. HUVECs: human umbilical vein endothelial cells.

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  Fig. 2 Activation of FOXO1 and inhibition of KLF2 under high glucose conditions. A: KLF2 and eNOS protein expression at the indicated concentration of glucose. B: serial immunoblot analysis of phosphorylated FOXO1 on serine 256 (p-FOXO1), total FOXO1 (t-FOXO1), and KLF2 expression in endothelial cells incubated under physiologically high glucose conditions (35 mM). C: quantification of triplicate experiments of A by densitometry. D: quantification of triplicate experiments of B by densitometry. *p<0.05 compared to the control, †p<0.05 compared to the value of 36 hours. KLF2: Krüppel-like factor 2, eNOS: endothelial nitric oxide synthase.

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  Fig. 3 Inhibition of shear flow-induced KLF2 expression under high glucose conditions. A: morphologic changes in HUVECs after shear stress application (24 hours, 12 dynes/cm2). B: immunoblot analysis evaluating the impact of shear stress and high glucose on the expression of KLF2 and eNOS. KLF2: Krüppel-like factor 2, HUVECs: human umbilical vein endothelial cells, eNOS: endothelial nitric oxide synthase.

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  Fig. 4 Suppression of KLF2 and eNOS by FOXO1 in endothelial cells. A: representative RT-PCR analysis figure evaluating blocking effect of FOXO1 by FOXO1 siRNA on KLF2 and eNOS mRNA expression in high glucose condition. B-D: quantification of triplicate experiments of A. *p<0.05 compared to the control. KLF2: Krüppel-like factor 2, eNOS: endothelial nitric oxide synthase, RT-PCR: reverse transcriptase-polymer chain reaction, siRNA: small interfering ribonucleic acid, mRNA: messenger ribonucleic acid.

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  Fig. 5 Suppression of KLF2 in the blood vessels from diabetic, OLEFT rats. Representative immunofluorescent microscopic images of carotid arteries stained with KLF2 (colored-red) in the OLETF rats (left) and the control LETO rats (right). KLF2: Krüppel-like factor 2, OLEFT: Otsuka Long Evans Tokushima Fatty, LETO: Long Evans Tokushima Otsuka.

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  Fig. 6 Comparison of KLF2 expression in endothelial cells at abdominal-celiac branch point between OLETF and control LETO rats. A: schematic showing the branch point of the celiac artery off the abdominal aorta. The line shows the section through which the samples were taken for immunohistochemical examination of KLF2 expression. B: representative photographs of immunofluorescent staining of blood vessels with KLF2 in the OLETF rats and the LETO rats. KLF2 expression is stained red at the branching point between the abdominal aorta and the celiac artery of the indicated rats. Arrow indicates 'atherosclerosis-resistant flow-dividing' side and 'atherosclerosis-prone lateral' side. C: semi-quantitative graph of KLF2-stained luminal circumferential length (4 rats in each group). KLF2: Krüppel-like factor 2, OLEFT: Otsuka Long Evans Tokushima Fatty, LETO: Long Evans Tokushima Otsuka.

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  Fig. 7 Evaluation of neointimal formation after induction of vascular remodeling. A: representative photomicrographs of common carotid arteries sections from the indicated rats 1 week after ligation. At the left are photographs from the sham-operated artery. At the right are photographs from the ligation-operated artery. Yellow arrow indicates intima. B: comparison of intimal thickness. n=8 (two slide sections in one carotid artery sample) in each group (4 rats in each group). Statistical comparisons between ligation-operated and sham-operated arteries were performed using a paired t-test for comparison for intra-group comparisons and a Student's t-test for inter-group comparisons. OLEFT: Otsuka Long Evans Tokushima Fatty, LETO: Long Evans Tokushima Otsuka.

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