Angptl4 Suppresses TNF- Stimulated Monocyte Adhesion to Endothelial Cells by Modulation of NFB Signaling

Abstract

Purpose: : Angiopoietin-like protein 4 (Angptl4) is a 70kD secreted glycoprotein, and a downstream target of the peroxisome proliferators-activated receptors alpha (PPARα) and gamma (PPARγ), which affect angiogenesis, metabolism, and inflammation. Previously, we reported that Angptl4 suppressed angiogenesis in the corneal micropocket assay and tumor xenograft model. In this study, we investigate the effect of Angptl4 in tumor necrosis factor alpha (TNF-α)-induced expression of adhesion molecules in endothelial cells.

Methods: : Human umbilical vein endothelial cells (HUVECs) at P4~7 were cultured with TNF-α (1 ng/ml) and angptl4 (0~3 µg/ml), and cellular proteins were extracted. Western blotting analysis was performed for intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), nuclear factor-ΚB (NFΚB), and phospho-NFΚB. Monocyte-endothelial cell adhesion assays were performed using HUVECs and DiI-labeled human monocytic U937 cells. After HUVECs were stimulated with TNF-α and angptl4 for 6 hours, DiI-labeled U937 cells were added with continued incubation for 1 hour. Non-adherent cells were removed by washing three times with pre-warmed PBS and monocytes adherent to HUVECs were counted.

Results: : TNF-α alone increased ICAM-1 and VCAM-1 protein levels in HUVECs after 6 hours; however, addition of Angptl4 suppressed ICAM-1 and VCAM-1 protein levels dose-dependently in western blot analysis. Moreover, Angptl4 reduced TNF-α-stimulated phospho-NFΚB protein induction as detected by western blot analysis. Monocyte-endothelial cell adhesion assays demonstrated that addition of Angptl4 reduced the adhesion of U937 to TNF-α-stimulated HUVECs.

Conclusions: : TNF-α-induced recruitment of monocytes may play an important role in the pathophysiology of ocular inflammatory disease. Our data suggest that Angptl4 suppresses monocyte adhesion to TNF-α-stimulated HUVECs by modulating NFΚB signaling. Therefore, Angptl4 represents an endogenously expressed protein that may have potential as a therapeutic agent for ocular inflammation.

Keywords: inflammation