Neutrophil elastase cleaves vascular endothelial growth factor to generate a fragment with altered activity.

摘要

Elastase released from neutrophils as a part of the innate immune system leads to both activation of tissue repair and inflammation. Excessive neutrophil elastase (NE) activity and altered vascular endothelial growth factor (VEGF) signaling have independently been implicated in the development and progression of pulmonary emphysema. In the present study we investigated the potential link between NE and VEGF. We noted that VEGF is a substrate for NE. Digestion of purified VEGF with NE generated a partially degraded disulfide linked fragment of VEGF. Mass spectrometric analysis revealed that NE likely cleaves VEGF at both the NH2 and COOH termini to produce VEGF fragment chains ∼5 kDa reduced in size. NE treatment of VEGF-laden endothelial cells as well as smooth muscle cells that endogenously express VEGF, generated VEGF fragments similar to those observed with purified VEGF. NE-generated VEGF fragment showed significantly reduced binding to VEGF receptor 2, heparin, fibronectin and neuropilin-1, yet retained the ability to bind to VEGF receptor 1. VEGF fragment also showed significantly reduced binding to smooth muscle cell-derived extracellular matrix compared to intact VEGF. Interestingly, VEGF fragment showed altered signaling in pulmonary artery endothelial cells compared with intact VEGF. Specifically, treatment with VEGF fragment did not activate extracellular-regulated kinases 1 and 2 (ERK1/2), yet resulted in activation of protein kinase B (Akt). Treatment of monocyte/macrophage RAW 264.7 cells with VEGF fragment, on the other hand, led to both Akt and ERK1/2 activation, increased VEGF receptor 1 expression, and stimulation of chemotaxis. A co-culture system was established to evaluate the role of NE released VEGF in recruiting macrophages and endothelial progenitor cells. High or low VEGF producing cells were co-cultured with macrophages, endothelial or endothelial progenitor cells and treated with NE. NE treatment stimulated macrophage and endothelial progenitor cell migration with the response being greater with the high VEGF expressing cells. However, NE treatment led to decreased endothelial cell migration due to VEGF cleavage to VEGF fragment. These findings suggest that the tissue response to NE-mediated injury might involve the generation of diffusible VEGF fragments that stimulate inflammatory cell recruitment and activation via VEGF receptor 1.