Transcription factor E4BP4 induces natural killer cell-derived inflammation and increases neointimal formation after vascular injury

Aim Adventitial inflammation is involved in the onset of Takayasu arteritis (TAK) and the formation of pathological conditions. E4BP4 (also known as NFIL3) is a basic leucine zipper (bZIP)-type transcription factor associated with circadian rhythms and cell viability, and previous in vitro studies have demonstrated that E4BP4 is essential for natural killer (NK) cell development and functional maturation. However, the specific role of E4BP4 in vascular inflammation in vivo remains unclear. Methods and Results Using E4BP4-deficient (E4BP4−/−) and wild-type (WT) mice, we investigated neointimal formation 7 and 14 days after femoral artery injury induced by an external vascular cuff model. Before injury, we observed no neointimal formation in the contralateral control femoral arteries of either E4BP4−/− or WT mice, and the vascular wall showed no structural differences, suggesting that vessel growth was normal in E4BP4−/− mice up to the age of 8–12 weeks. Quantitative PCR revealed that mRNA levels of E4BP4 (3.4-fold, P < 0.001) significantly increased in cuffed arteries compared with control arteries in WT mice at 3 days after injury (Fig. A). As expected, E4BP4 mRNA was not detected in E4BP4−/− mice. Immunohistochemical analysis in WT mice revealed that E4BP4 protein predominantly localized in the inflammatory cells of the adventitia at 7 days post-injury (Fig. B, left), and double staining demonstrated that most of the inflammatory cells were NK cells (Fig. C). At 14 days post-injury, E4BP4 was expressed throughout the entire vascular wall, including endothelial cells, smooth muscle cells of the media, and inflammatory cells (Fig. B, right). These results indicate that cuff injury induces E4BP4 mRNA expression, leading to the accumulation of E4BP4-positive NK cells in the adventitia and subsequent expression of E4BP4 throughout the vascular wall at 14 days post-injury in WT mice. Furthermore, the positive areas of IL-6 (P < 0.05), TNF-α (P < 0.05), and IFN-γ (P < 0.05) in the intima of E4BP4−/− mice were significantly smaller than those of WT mice. Finally, the mean intimal area and the intima/media ratio of E4BP4−/− mice decreased by 86% and 97%, respectively, compared with WT mice. Our findings indicate that E4BP4 might induce vascular inflammation following vascular injury. Conclusions These results indicate that E4BP4 deficiency attenuates NK cell-derived inflammation and reduces inflammatory cytokine expression, thereby reducing intimal thickening after injury. The present study is the first to demonstrate that E4BP4 plays an important role in the promotion of vascular inflammation after injury in vivo, suggesting that targeting E4BP4 may represent a novel therapeutic strategy to inhibit vascular inflammation, such as TAK.