Interferon Regulatory Factor 4 Inhibits Neointima Formation by Engaging Krüppel-Like Factor 4 Signaling

Wen-Lin Cheng; Zhi-Gang She; Juan-Juan Qin; Jun-Hong Guo; Fu-Han Gong; Peng Zhang; Chun Fang; Song Tian; Xue-Yong Zhu; Jun Gong; Zhi-Hua Wang; Zan Huang; Hongliang Li

doi:10.1161/CIRCULATIONAHA.116.026046

Interferon Regulatory Factor 4 Inhibits Neointima Formation by Engaging Krüppel-Like Factor 4 Signaling

Circulation. 2017 Oct 10;136(15):1412-1433. doi: 10.1161/CIRCULATIONAHA.116.026046. Epub 2017 Aug 29.

Authors

Affiliations

¹ From Department of Cardiology, Renmin Hospital of Wuhan University, China (W.-L.C., Z.-G.S., F.-H.G., S.T., X.-Y.Z., Z.-H.W., H.L.); Basic Medical School (W.-L.C., Z.-G.S., J.-J.Q., J.-H.G., F.-H.G., P.Z., C.F., S.T., X.-Y.Z., J.G., Z.-H.W., Z.H., H.L.), Medical Research Institute, School of Medicine (W.-L.C., Z.-G.S., J.-J.Q., J.-H.G., F.-H.G., P.Z., C.F., S.T., X.-Y.Z., J.G., Z.-H.W., Z.H., H.L.), and College of Life Science (J.G., Z.H.), Wuhan University, China; and Institute of Model Animal of Wuhan University, China (W.-L.C., Z.-G.S., J.-J.Q., J.-H.G., F.-H.G., P.Z., C.F., S.T., X.-Y.Z., J.G., Z.-H.W., Z.H., H.L.).
² From Department of Cardiology, Renmin Hospital of Wuhan University, China (W.-L.C., Z.-G.S., F.-H.G., S.T., X.-Y.Z., Z.-H.W., H.L.); Basic Medical School (W.-L.C., Z.-G.S., J.-J.Q., J.-H.G., F.-H.G., P.Z., C.F., S.T., X.-Y.Z., J.G., Z.-H.W., Z.H., H.L.), Medical Research Institute, School of Medicine (W.-L.C., Z.-G.S., J.-J.Q., J.-H.G., F.-H.G., P.Z., C.F., S.T., X.-Y.Z., J.G., Z.-H.W., Z.H., H.L.), and College of Life Science (J.G., Z.H.), Wuhan University, China; and Institute of Model Animal of Wuhan University, China (W.-L.C., Z.-G.S., J.-J.Q., J.-H.G., F.-H.G., P.Z., C.F., S.T., X.-Y.Z., J.G., Z.-H.W., Z.H., H.L.). lihl@whu.edu.cn.

PMID: 28851732
DOI: 10.1161/CIRCULATIONAHA.116.026046

Abstract

Background: The mechanisms underlying neointima formation remain unclear. Interferon regulatory factors (IRFs), which are key innate immune regulators, play important roles in cardiometabolic diseases. However, the function of IRF4 in arterial restenosis is unknown.

Methods: IRF4 expression was first detected in human and mouse restenotic arteries. Then, the effects of IRF4 on neointima formation were evaluated with universal IRF4-deficient mouse and rat carotid artery injury models. We performed immunostaining to identify IRF4-expressing cells in the lesions. Smooth muscle cell (SMC)-specific IRF4-knockout (KO) and -transgenic (TG) mice were generated to evaluate the effects of SMC-IRF4 on neointima formation. We used microarray, bioinformatics analysis, and chromatin immunoprecipitation assay to identify the downstream signals of IRF4 and to verify the targets in vitro. We compared SMC-IRF4-KO/Krüppel-like factor 4 (KLF4)-TG mice with SMC-IRF4-KO mice and SMC-specific IRF4-TG/KLF4-KO mice with SMC-specific IRF4-TG mice to investigate whether the effect of IRF4 on neointima formation is KLF4-dependent. The effect of IRF4 on SMC phenotype switching was also evaluated.

Results: IRF4 expression in both the human and mouse restenotic arteries is eventually downregulated. Universal IRF4 ablation potentiates neointima formation in both mice and rats. Immunostaining indicated that IRF4 was expressed primarily in SMCs in restenotic arteries. After injury, SMC-IRF4-KO mice developed a thicker neointima than control mice. This change was accompanied by increased SMC proliferation and migration. However, SMC-specific IRF4-TG mice exhibited the opposite phenotype, demonstrating that IRF4 exerts protective effects against neointima formation. The mechanistic study indicated that IRF4 promotes KLF4 expression by directly binding to its promoter. Genetic overexpression of KLF4 in SMCs largely reversed the neointima-promoting effect of IRF4 ablation, whereas ablation of KLF4 abolished the protective function of IRF4, indicating that the protective effects of IRF4 against neointima formation are KLF4-dependent. In addition, IRF4 promoted SMC dedifferentiation.

Conclusions: IRF4 protects arteries against neointima formation by promoting the expression of KLF4 by directly binding to its promoter. Our findings suggest that this previously undiscovered IRF4-KLF4 axis plays a key role in vasculoproliferative pathology and may be a promising therapeutic target for the treatment of arterial restenosis.

Keywords: Kruppel-like transcription factors; cell proliferation; coronary restenosis; interferon regulatory factor-4; smooth muscle cell.

MeSH terms

Animals
Gene Expression Regulation*
Humans
Interferon Regulatory Factors* / genetics
Interferon Regulatory Factors* / metabolism
Kruppel-Like Factor 4
Kruppel-Like Transcription Factors* / genetics
Kruppel-Like Transcription Factors* / metabolism
Mice
Mice, Knockout
Muscle, Smooth, Vascular* / metabolism
Muscle, Smooth, Vascular* / pathology
Neointima* / genetics
Neointima* / metabolism
Neointima* / pathology
Oligonucleotide Array Sequence Analysis
Rats

Substances

Interferon Regulatory Factors
KLF4 protein, human
Klf4 protein, mouse
Klf4 protein, rat
Kruppel-Like Factor 4
Kruppel-Like Transcription Factors
interferon regulatory factor-4