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.

EGFL7: a unique angiogenic signaling factor in vascular development and disease

“…61 Specifically, Egfl7 overexpression in breast and lung carcinoma cells enhances tumor progression in immunocompetent mice and appears to do so by reducing the expression of tumor endothelial leukocyte adhesion molecules, including ICAM-1 and VCAM-1, thus preventing host immune cell infiltration. 61 The repression of ICAM-1 expression by EGFL7 is in keeping with observations made during endothelial cell injury 33,35 and highlights a possible novel mechanism by which EGFL7 promotes tumorigenesis.…”

“…34 EGFL7 may also stimulate vascular regeneration by repressing key steps in the inflammatory activation of endothelial cells. 33,35 Specifically, it has been shown that EGFL7 represses NFB activation and the subsequent expression of ICAM-1 in response to hypoxia/reoxygenationinduced and calcineurin inhibition-induced endothelial injury. 33,35 In contrast to hypoxic conditions, EGFL7 expression is inhibited after hyperoxic exposure in both neonatal lungs in vivo and in endothelial cells in vitro, and reduced levels are associated with endothelial cell death.…”

“…33,35 Specifically, it has been shown that EGFL7 represses NFB activation and the subsequent expression of ICAM-1 in response to hypoxia/reoxygenationinduced and calcineurin inhibition-induced endothelial injury. 33,35 In contrast to hypoxic conditions, EGFL7 expression is inhibited after hyperoxic exposure in both neonatal lungs in vivo and in endothelial cells in vitro, and reduced levels are associated with endothelial cell death. 36 In support, EGFL7 overexpression in endothelial cell lines results in a protective effect against hyperoxiainduced apoptosis by preventing cytochrome c release, inhibiting caspase-3 activation, and preventing the induction of BAX expression, all of which are hallmarks of mitochondria-induced apoptosis.…”

Endothelial Cell Activation Is Regulated by Epidermal Growth Factor-like Domain 7 (Egfl7) during Inflammation

“…Previously, Badiwala et al (10) reported that treating human coronary artery endothelial cells stimulated by cyclosporin A or tacrolimus with exogenously added recombinant Egfl7 repressed neutrophil adhesion to these cells, decreased NF-B DNA-binding, and lowered ICAM-1 expression. We had also reported earlier that an Egfl7-producing tumor cell-conditioned medium prevented the adhesion of T-cells to the endothelial cells (7).…”

Effects of Immunosuppressive agents on neutrophils inflammatory response in humans: An inflammatory perspective on coronary allograft vasculopathy

“…Indeed while CNIs can prevent acute rejection very effectively, they do not seem to have any significant effect on the prevention of CAV [10] , indicating that CAV is mediated via some pathways not modulated via the calcineurin pathway Badiwala et al [61] demonstrated that CsA induces injuries by increasing ICAM-1 expression on human coronary artery endothelial cells, thus promoting neutrophil adhesion. This effect could ultimately increase local cardiac inflammation and consequently facilitate the development of CAV.…”