Abstract
Purpose: :
Neutrophil elastase (NE) contributes to tissue destruction and scar formation at sites of injury and inflammation. The angiogenic factor VEGF also mediates the tissue response to injury and inflammation. While vascular endothelial cells are generally considered to be the principal target of VEGF, recent studies indicate that other cells including monocytes and macrophages also respond to VEGF through VEGF-R1. The purpose of this study was to test the hypothesis that NE-mediated cleavage of VEGF alters its activity.
Methods: :
Human NE and recombinant VEGF were allowed to react in vitro and VEGF digestion was analyzed by SDS-PAGE and mass spectrometry. The activity of NE-cleaved VEGF fragments (VEGFf) was evaluated in bovine aortic endothelial cells and the mouse mononcyte/macrophage cell line RAW264.7 using western blots to evaluate signaling, and in vitro assays to measure cell proliferation and migration. VEGFf binding to VEGF receptors, fibronectin and heparin was evaluated using cell-free binding assays. VEGF release from cell cultures was quantified by ELISA.
Results: :
Digestion of VEGF165 with NE generated a partially degraded disulfide linked fragment (VEGFf). Mass spectrometric analysis revealed that NE cleaves VEGF165 at both the N- and C-termini. NE treatment of VEGF-expressing smooth muscle cells led to the release of VEGFf. NE-generated VEGFf showed reduced binding to VEGF-R2, heparin, and fibronectin, yet retained the ability to bind to VEGF-R1. VEGFf did not activate Erk1/2 in endothelial cells, yet resulted in enhanced activation of Akt, and facilitated cell survival when added with intact VEGF. Treatment of RAW264.7 cells with VEGFf, on the other hand, led to both Akt and ERK1/2 activation, increased VEGFR1 expression and stimulated chemotaxis and proliferation.
Conclusions: :
The tissue response to NE-mediated injury and inflammation in the eye might involve the generation of diffusible VEGFf that stimulates inflammatory cell recruitment and activation via VEGF-R1.
Keywords: inflammation • vascular endothelial growth factor • extracellular matrix