Epidermal Growth Factor–Like Domain 7 Is a Novel Inhibitor of Neutrophil Adhesion to Coronary Artery Endothelial Cells Injured by Calcineurin Inhibition

Abstract

Background— We investigated the effect of epidermal growth factor–like domain 7 (Egfl7) on nuclear factor-κB activation, intercellular adhesion molecule-1 expression, and neutrophil adhesion to human coronary artery endothelial cells after calcineurin-inhibition–induced injury. Methods and Results— Human coronary endothelial cells were incubated with cyclosporine (CyA) 10 μg/mL with or without Egfl7 (100 ng/mL) or the Notch receptor activator Jagged1 (200 ng/mL) for 6 to 48 hours. CyA upregulated nuclear factor-κB (p65) activity (128±2% of control, P <0.001) in nuclear extracts, as determined with a DNA-binding activity ELISA. This activity was inhibited by Egfl7 (86±3% of control; P <0.001 versus CyA alone). Jagged1 blocked Egfl7-induced nuclear factor-κB inhibition (105±4% of control; P <0.05 versus CyA plus Egfl7). CyA upregulated cell-surface intercellular adhesion molecule-1 expression (215±13% of control; P <0.001), as determined by flow cytometry. This expression was suppressed by Egfl7 (148±5%; P <0.001 versus CyA alone). Jagged1 attenuated the intercellular adhesion molecule-1–suppressive effect of Egfl7 when administered with CyA (193±3% versus 148±5%; P <0.01). CyA increased neutrophil adhesion to human coronary endothelial cells (control 20±5%, CyA 37±3%; P <0.001 versus control) in a nonstatic neutrophil adhesion assay. This increase was attenuated by Egfl7 (22±6%; P <0.001 versus CyA alone). Jagged 1 attenuated the effect of Egfl7 on neutrophil adhesion (31±3%; P <0.001 versus Egfl7 plus CyA). Conclusions— Our study reveals that Egfl7 is a potent inhibitor of neutrophil adhesion to human coronary endothelial cells subsequent to calcineurin-inhibition–induced injury. Mechanistically, Egfl7 blocked nuclear factor-κB pathway activation and intercellular adhesion molecule-1 expression, which suggests that it may have significant antiinflammatory properties. Because Jagged1 blocked the effect of Egfl7, Notch receptor antagonism may contribute to the mechanism of action of Egfl7.