MicroRNA-181b regulates NF-κB–mediated vascular inflammation
Xinghui Sun, Basak Icli, Akm Khyrul Wara, Nathan Belkin, Shaolin He, Lester Kobzik, Gary M. Hunninghake, Miguel Pinilla Vera, MICU Registry, Timothy S. Blackwell, Rebecca M. Baron, Mark W. Feinberg
Xinghui Sun, Basak Icli, Akm Khyrul Wara, Nathan Belkin, Shaolin He, Lester Kobzik, Gary M. Hunninghake, Miguel Pinilla Vera, MICU Registry, Timothy S. Blackwell, Rebecca M. Baron, Mark W. Feinberg
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Research Article

MicroRNA-181b regulates NF-κB–mediated vascular inflammation

Abstract

EC activation and dysfunction have been linked to a variety of vascular inflammatory disease states. The function of microRNAs (miRNAs) in vascular EC activation and inflammation remains poorly understood. Herein, we report that microRNA-181b (miR-181b) serves as a potent regulator of downstream NF-κB signaling in the vascular endothelium by targeting importin-α3, a protein that is required for nuclear translocation of NF-κB. Overexpression of miR-181b inhibited importin-α3 expression and an enriched set of NF-κB–responsive genes such as adhesion molecules VCAM-1 and E-selectin in ECs in vitro and in vivo. In addition, treatment of mice with proinflammatory stimuli reduced miR-181b expression. Rescue of miR-181b levels by systemic administration of miR-181b “mimics” reduced downstream NF-κB signaling and leukocyte influx in the vascular endothelium and decreased lung injury and mortality in endotoxemic mice. In contrast, miR-181b inhibition exacerbated endotoxin-induced NF-κB activity, leukocyte influx, and lung injury. Finally, we observed that critically ill patients with sepsis had reduced levels of miR-181b compared with control intensive care unit (ICU) subjects. Collectively, these findings demonstrate that miR-181b regulates NF-κB–mediated EC activation and vascular inflammation in response to proinflammatory stimuli and that rescue of miR-181b expression could provide a new target for antiinflammatory therapy and critical illness.

Authors

Xinghui Sun, Basak Icli, Akm Khyrul Wara, Nathan Belkin, Shaolin He, Lester Kobzik, Gary M. Hunninghake, Miguel Pinilla Vera, Timothy S. Blackwell, Rebecca M. Baron, Mark W. Feinberg

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Figure 3

miR-181b inhibits the activation of the NF-κB signaling pathway.

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miR-181b inhibits the activation of the NF-κB signaling pathway. (A) Luc...
(A) Luciferase activity of reporters containing either the NF-κB concatemer or VCAM-1 promoter in HUVECs transfected with miRNA negative control or miR-181b mimics and miRNA inhibitor negative control or miR-181b inhibitor after 12 hours treatment with 10 ng/ml of TNF-α. #P < 0.05; *P < 0.01. Values represent mean ± SD; n = 3. (B) p65 staining in HUVECs transfected with miRNA negative control or miR-181b mimics. Thirty-six hours after transfection, cells were treated with 10 ng/ml TNF-α for 60 minutes and processed for immunostaining with antibodies against p65. Cells were stained with DAPI to visualize the nucleus. Cy3-conjugated miRNA negative control was transfected at 3 nM concentration to show transfection efficiency in both groups. Images were acquired by confocal microscopy from 3 independent experiments, and values represent mean ± SD. *P < 0.01. Scale bars: 20 μm. (C) The indicated proteins were detected in cytoplasmic or nuclear fractions prepared from HUVECs transfected with miRNA negative control or miR-181b mimics and treated with 10 ng/ml TNF-α for 1 hour. Densitometry was performed and fold change of p65 and p50 protein expression after normalization is shown below the corresponding band. Quantifications from 3 independent experiments were shown in D, and values represent mean ± SD. *P < 0.05. (E) Western blot analysis of phosphorylated IκBα or IKKβ in HUVECs transfected with miRNA negative control, or miR-181b mimics and treated with 10 μM MG-132 or DMSO for 2 hours followed by 10 ng/ml TNF-α for the indicated times.