Kruppel-like factor 2 regulates endothelial barrier function

Abstract

Objective: A central function of the endothelium is to serve as a selective barrier that regulates fluid and solute exchange. Although perturbation of barrier function can contribute to numerous disease states, our understanding of the molecular mechanisms regulating this aspect of endothelial biology remains incompletely understood. Accumulating evidence implicates the Kruppel-like factor 2 (KLF2) as a key regulator of endothelial function. However, its role in vascular barrier function is unknown.

Methods and results: To assess the role of KLF2 in vascular barrier function in vivo, we measured the leakage of Evans blue dye into interstitial tissues of the mouse ear after treatment with mustard oil. By comparison with KLF2(+/+) mice, KLF2(+/-) mice exhibited a significantly higher degree of vascular leak. In accordance with our in vivo observation, adenoviral overexpression of KLF2 in human umbilical vein endothelial cells strongly attenuated the increase of endothelial leakage by thrombin and H(2)O(2) as measured by fluorescein isothiocyanate dextrans (FITC-dextran) passage. Conversely, KLF2 deficiency in human umbilical vein endothelial cells and primary endothelial cells derived from KLF2(+/-) mice exhibited a marked increase in thrombin and H(2)O(2)-induced permeability. Mechanistically, our studies indicate that KLF2 confers barrier-protection via differential effects on the expression of key junction protein occludin and modification of a signaling molecule (myosin light chain) that regulate endothelial barrier integrity.

Conclusions: These observations identify KLF2 as a novel transcriptional regulator of vascular barrier function.

Figure 1

Hemizygous deficiency of KLF2 shows increased vascular permeability in response to Mustard oil. (A) Seven to eight weeks old male wild type and KLF2 heterozygous mice were used for this experiment. Evans blue dye was injected into the mice through internal jugular vein. Mustard oil was applied 1 minute later to the mice ear, photographs were taken after 15 minutes; then the mice were sacrificed and ear lobes harvested. Representative pictures are shown. (B) The ears were later cut into small pieces and the dye extracted using 1 ml of formamide, the EBD amount in each sample was quantified by spectrophotometer at a wavelength of 620nm and normalized to ear weight. In KLF2 heterozygous mice the amount of dye extravasated was statistically significant higher when compared to that of wild type (n=9, p<0.05).

Figure 2

Overexpression of KLF2 decreases endothelial leakage. (A) KLF2 overexpressed cells significantly attenuates thrombin mediated inter-endothelial gap formation when compared to Ad-GFP infected cells. Confluent HUVEC were infected with control (Ad-GFP) or KLF2 adenovirus for 48 hrs, exposed to thrombin (1U/ml for 15 minutes), and inter-endothelial gap formation assessed by actin staining. Percentage of exposed areas was quantified by NIH Image J software. The control group (Ad-GFP with thrombin) was normalized to 100%. A representative of three independent experiments is shown. Scale bar, 50 μm. (B) KLF2 decreases endothelial leakage when HUVEC were challenged with thrombin (1U/ml). HUVEC were infected as in (A), treated with thrombin (1U/ml), and leakage assay was performed to measure FITC-Dextran passage. N=6, p<0.01. (C) KLF2 decreases endothelial leakage when HUVEC were treated with 200uM of tert-butyl hydrogen peroxide. N=6, p<0.01.

Figure 3

KLF2 deficiency increases endothelial leakage. (A) KLF2 deficiency in HUVEC lead to an enhancement of inter-endothelial gap formation response to thrombin treatment. HUVEC were transfected with control siRNA and KLF2 siRNA, five days after transfection, cells were treated with thrombin (1U/ml) for gap formation assessment by actin staining. Percentage of exposed areas was quantified by NIH Image J software. The control group (NS with thrombin) was normalized to 100%. A representative of three independent experiments is shown. Scale bar, 50 μm. (B) Deficiency of KLF2 lead to increased endothelial leakage in response to thrombin. HUVEC were transfected with control siRNA and KLF2 siRNA for two days, then cells were replated onto transwell; permeability analysis was performed following treatment with thrombin. N=6, p<0.01. (C) KLF2 deficiency leads to increased endothelial leakage in response to hydrogen peroxide. HUVEC were transfected as above, and treated with hydrogen peroxide (200uM) followed by permeability assay. N=6, p<0.01. (D) Partial deficiency of KLF2 in primary ECs lead to a significant increase of endothelial leakage when treated with thrombin. Confluent lung microvascular endothelial cells from KLF2^+/+ and KLF2^+/− mice were treated with thrombin, then permeability assay performed. N=6, p<0.01.

Figure 4

KLF2 increases the expression of tight junction protein occludin. (A) siRNA-mediated KLF2 knockdown reduces occludin. HUVEC were transfected with non-specific siRNA and KLF2 siRNA for 48 hrs, and protein harvested for Western analysis. Representative blots of three independent experiments are shown. (B) Partial KLF2 deficiency in primary ECs results in decrease of occludin expression. Primary microvascular ECs were isolated from KLF2^+/+ and KLF2^+/− mice, extracted total protein were subjected to western blot. WT = KLF2^+/+, Het = KLF2^+/−. (C) Overexpression of KLF2 increases occludin expression. HUVEC were infected with Ad-GFP and KLF2 adenovirus for 24 hrs. Cells were treated with thrombin (1U/ml) for 15 and 30 minutes and proteins extracted for western blot analysis. Representative blots of three independent experiments are shown.

Figure 5

KLF2 inhibits phosphorylation of MLC in response to agonists. (A) HUVEC were infected with Ad-GFP and KLF2 adenovirus for 24 hrs, cells were then treated with thrombin (1U/ml) for 15 and 30 minutes, proteins were then extracted. Western blot analysis reveals attenuation of MLC phosphorylation in KLF2 overexpressed cells. Representative blot of three independent experiments is shown. (B) HUVEC were infected as above, followed by treatment with hydrogen peroxide for 30 minutes and 60 minute, total protein were isolated and subjected to western blot. Representative blots of three independent experiments are shown. (C) HUVEC infected with Ad-GFP and KLF2 adenovirus for 24 hrs were treated with thrombin (1U/ml) for 15 minutes, then immunostaining with phospho-MLC antibody was performed. Representative pictures of three independent experiments are shown. Scale bar, 50 μm. (D) HUVEC were transfected with non specific siRNA and siKLF2 using siPortAmine. After 48 hrs the cells were treated with thrombin (1U/ml) for 1 minute and total protein harvested using RIPA buffer. Western blot analysis revealed increased phosphorylation of MLC in siKLF2 transfected cells when compared to non specific siRNA transfected cells. (E) Primary lung microvascular endothelial cells were isolated from KLF2^+/+ and KLF2^+/− mice, they were then treated with mouse thrombin for 1 minute, proteins were extracted and western blot performed. A significant increase of MLC phosphorylation was observed in KLF2^+/− ECs than that of KLF2^+/+ ECs. A representative blot of three independent experiments is shown.

Figure 5

KLF2 inhibits phosphorylation of MLC in response to agonists. (A) HUVEC were infected with Ad-GFP and KLF2 adenovirus for 24 hrs, cells were then treated with thrombin (1U/ml) for 15 and 30 minutes, proteins were then extracted. Western blot analysis reveals attenuation of MLC phosphorylation in KLF2 overexpressed cells. Representative blot of three independent experiments is shown. (B) HUVEC were infected as above, followed by treatment with hydrogen peroxide for 30 minutes and 60 minute, total protein were isolated and subjected to western blot. Representative blots of three independent experiments are shown. (C) HUVEC infected with Ad-GFP and KLF2 adenovirus for 24 hrs were treated with thrombin (1U/ml) for 15 minutes, then immunostaining with phospho-MLC antibody was performed. Representative pictures of three independent experiments are shown. Scale bar, 50 μm. (D) HUVEC were transfected with non specific siRNA and siKLF2 using siPortAmine. After 48 hrs the cells were treated with thrombin (1U/ml) for 1 minute and total protein harvested using RIPA buffer. Western blot analysis revealed increased phosphorylation of MLC in siKLF2 transfected cells when compared to non specific siRNA transfected cells. (E) Primary lung microvascular endothelial cells were isolated from KLF2^+/+ and KLF2^+/− mice, they were then treated with mouse thrombin for 1 minute, proteins were extracted and western blot performed. A significant increase of MLC phosphorylation was observed in KLF2^+/− ECs than that of KLF2^+/+ ECs. A representative blot of three independent experiments is shown.