Background-Epidermal growth factor-like domain 7 (Egfl7) is a chemoattractant for endothelial cells, and its expression is restricted to endothelial cells. Hypoxia/reoxygenation (H/R) induced endothelial injury that occurs during transplantation contributes to the subsequent development of allograft vasculopathy. We investigated the effect of Egfl7 on endothelial cell intercellular adhesion molecule 1 expression in response to H/R injury. Methods and Results-Human coronary artery endothelial cells were submitted to hypoxia (0.1% O 2 ) followed by normoxia (21% O 2 ) in the presence or absence of Egfl7 (100 ng/mL). Hypoxia alone increased the expression of Egfl7ϫ140Ϯ8% of control at 3 hours (nϭ6; PϽ0.05) and 385Ϯ50% of control at 6 hours (nϭ6; PϽ0.001). Incubation with Egfl7 during the reoxygenation period prevented intercellular adhesion molecule 1 upregulation (mean fluorescence intensity: 5.37Ϯ0.92 versus 3.81Ϯ0.21; PϽ0.05; nϭ4 per group). Nuclear factor-B nuclear localization on H/R injury was blocked by Egfl7 administration (cytosolic/nuclear ratio of 0.93Ϯ0.01 versus 1.44Ϯ0.24; PϽ0.05; nϭ4 per group). Inhibitor of nuclear factor-B protein level was significantly reduced on H/R injury (26Ϯ4.6% of control expression; PϽ0.05; nϭ4 per group); however, concurrent incubation with Egfl7 attenuated this reduction (46Ϯ6.2% of control expression; PϽ0.05 when compared with H/R injury alone; nϭ4 per group). Conclusions-Our study reveals the novel observation that hypoxia upregulates human coronary artery endothelial cells expression of Egfl7 and that Egfl7 inhibits expression of intercellular adhesion molecule 1 subsequent to H/R injury. Mechanistically, Egfl7 prevented nuclear factor-B nuclear localization and augmented inhibitor of nuclear factor-B protein levels, suggesting that it inhibits nuclear factor-B activation, a key step in the inflammatory activation of endothelial cells. Egfl7 may be protective against H/R injury incurred during transplantation and may modulate the events that lead to the development of graft vasculopathy. (Circulation. 2010;122[suppl 1]:S156 -S161.)

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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.

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