Abstract 14663: Epidermal Growth Factor-Like Domain 7 is a Novel Inhibitor of Neutrophil Adhesion to Coronary Artery Endothelial Cells Injured by Calcineurin Inhibition
Background: Neutrophil adhesion is central to the inflammatory cascade that occurs following endothelial injury. We investigated the effect of epidermal growth factor-like domain 7 (Egfl7) on NFkB activation, ICAM-1 expression and neutrophil adhesion to human coronary endothelial cells (HCAEC) following calcineurin inhibition induced injury.
Methods: HCAEC (n=4 to 16) were incubated with cyclosporine (CyA) 10 μg/mL or 0.01% DMSO (control) +/− Egfl7 (100 ng/mL) or the NOTCH receptor activator Jagged1 (200 ng/mL) for 6 to 48h. NFkB (p65) activity in nuclear extracts was determined using a DNA binding activity ELISA. Cell surface expression of ICAM-1 was determined by flow cytometry. A non-static neutrophil adhesion assay was performed using neutrophils activated with PMA (10 ng/mL).
Results: CyA up-regulated NFkB activity (128±2% of control, p<0.001) which was inhibited by co-treatment with Egfl7 (86±3% of control, p<0.001 vs. CyA alone). Jagged1 blocked Egfl7 induced NFkB inhibition (105±4% of control, p<0.05 vs. CyA+Egfl7). CyA up-regulated cell surface ICAM-1 expression (215±13% of control, p<0.001), which was suppressed by co-treatment with Egfl7 (148±5%, p=0.004 vs. CyA alone). Jagged1 attenuated the ICAM-1 suppressive effect of Egfl7 when administered in combination with CyA (193±3% vs. 148±5%, p=0.006). CyA significantly increased neutrophil adhesion to HCAEC (Control:20±5%; CyA:37±3%, p<0.001 vs. control), which was attenuated by co-treatment with Egfl7 (22±6%, p<0.001 vs. CyA alone). Jagged 1 attenuated the effect of Egfl7 on neutrophil adhesion (31±3%, p<0.001 vs. Egfl7+CyA).
Conclusions: Our study reveals the novel observation that Egfl7 is a potent inhibitor of neutrophil adhesion to HCAECs subsequent to CyA induced injury. Mechanistically, Egfl7 blocked NFkB pathway activation and ICAM-1 expression, suggesting that it has significant anti-inflammatory properties. Since Jagged1 blocked the effect of Egfl7, NOTCH receptor antagonism may contribute to the mechanism of action of Egfl7. Thus, we conclude that Egfl7 may be protective against calcineurin inhibition induced endothelial injury incurred after cardiac transplantation, and thus may modulate events that lead to the development of cardiac allograft vasculopathy.
- © 2010 by American Heart Association, Inc.