IRF2BP2 prevents ox‑LDL‑induced inflammation and EMT in endothelial cells via regulation of KLF2

Figures

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  Figure 1. Protective effects of IRF2BP2 against ox-LDL-induced inflammation and EMT. (A) HUVECs were transfected with empty vector or pcDNA3.1-IRF2BP2. The mRNA expression levels of IRF2BP2 were measured using reverse transcription-quantitative PCR analysis. ***P<0.001 vs. the vector group. (B) The protein expression levels of IRF2BP2 were determined by western blot analysis. **P<0.01 vs. the vector group. (C) Following transfection, HUVECs were treated with ox-LDL for 24 h. The cell viability in different groups was determined using a Cell Counting Kit-8 assay. (D) The levels of TNF-α, IL-1β and IL-6 in the culture medium were assessed using ELISA kits. (E) The protein expression levels of VE-cadherin and vimentin were evaluated using western blot analysis. **P<0.01, ***P<0.001 vs. the control group; #P<0.01, ##P<0.05 and ###P<0.001 vs. the ox-LDL + vector group. IRF2BP2, interferon regulatory factor binding protein 2; ox-LDL, oxidized low-density lipoprotein; HUVECs, human umbilical vein endothelial cells; TNF-α, tumor necrosis factor α; IL-1β, interleukin 1β.

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  Figure 2. IRF2BP2 increases the expression of KLF2 and improves endothelial function. (A) The expression levels of ICAM-1 and VCAM-1 were determined using reverse transcription-quantitative PCR analysis. (B) The protein expression levels of KLF2, p-eNOS, total eNOS and GAPDH were measured using western blot analysis. (C) A Nitrate/Nitrite assay kit was utilized to estimate the release of NO. (D) The expression of KLF2 was evaluated by immunofluorescence analysis (magnification, x200). Scale bar, 50 µm. **P<0.01, ***P<0.001 vs. the control group; ##P<0.05, ###P<0.001 vs. the ox-LDL + vector group. IRF2BP2, interferon regulatory factor binding protein 2; KLF2, Krüppel-like factor 2; ICAM-1, intercellular adhesion molecule 1; VCAM-1, vascular cell adhesion molecule 1; p-eNOS, phosphorylated endothelial nitric oxide synthase; NO, nitric oxide; ox-LDL, oxidized low-density lipoprotein.

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  Figure 3. IRF2BP2 attenuates inflammation and EMT via KLF2. (A) The interaction between IRF2BP2 and KLF2 was confirmed using a Co-IP assay. (B) HUVECs were transfected with scrambled siRNA, siKLF2#1 or siKLF2#2. The mRNA expression levels of KLF2 were measured using reverse transcription-quantitative PCR analysis. ***P<0.001 vs. the scramble group. (C) The protein expression levels of KLF2 were evaluated using western blot analysis. **P<0.01, ***P<0.001 vs. the scrambled group. (D) The cell viability in different groups was determined using a CCK-8 assay. (E) The levels of TNF-α, IL-1β and IL-6 in the culture medium were assessed using ELISA kits. (F) The protein expression levels of VE-cadherin and vimentin were evaluated using western blot analysis. ***P<0.001 vs. the control group; ##P<0.05, ###P<0.001 vs. the ox-LDL group; ΔP<0.05, ΔΔP<0.01, ΔΔΔP<0.001 vs. the ox-LDL + IRF2BP2 + scramble group. IRF2BP2, interferon regulatory factor binding protein 2; KLF2, Krüppel-like factor 2; EMT, endothelial-to-mesenchymal transition; HUVECs, human umbilical vein endothelial cells; siRNA, small interfering RNA; TNF-α, tumor necrosis factor α; IL-1β, interleukin 1β; ox-LDL, oxidized low-density…

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  Figure 4. KLF2 is required for IRF2BP2-regulated endothelial function. (A) The mRNA expression levels of ICAM-1 and VCAM-1 were measured by reverse transcription-quantitative PCR analysis. (B) The protein expression levels of total eNOS, p-eNOS and GAPDH were evaluated using western blot analysis. (C) A Nitrate/Nitrite assay kit was used to estimate the release of NO. ***P<0.001 vs. the control group; ##P<0.05, ###P<0.001 vs. the ox-LDL group; ΔP<0.05 vs. the ox-LDL + IRF2BP2 + scrambled group. KLF2, Krüppel-like factor 2; IRF2BP2, interferon regulatory factor binding protein 2; ICAM-1, intercellular adhesion molecule 1; VCAM-1, vascular cell adhesion molecule 1; p-eNOS, phosphorylated endothelial nitric oxide synthase; NO, nitric oxide; ox-LDL, oxidized low-density lipoprotein.