Proinflammatory mediators stimulate neutrophil-directed angiogenesis

Background: Vascular endothelial growth factor (VEGF; vascular permeability factor) is one of the most potent proangiogenic cytokines, and it plays a central role in mediating the process of angiogenesis or new blood vessel f ormation. Neutrophils (PMNs) recently have been shown to produce VEGF. Hypothesis: The acute inflammatory response is a potent stimulus for PMN-di rected angiogenesis. Methods: Neutrophils were isolated from healthy volunteers and stimulated w ith lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-alpha), inte rleukin 6 (IL-6), and anti-human Fas monoclonal antibody. Culture supernata nts were assayed for VEGF using enzyme-linked immunosorbent assays. Culture supernatants from LPS- and TNF-alpha-stimulated PMNs were then added to hu man umbilical vein endothelial cells and human microvessel endothelial cell s and assessed for endothelial cell proliferation using 5-bromodeoxyuridine labeling. Tubule formation was also assessed on MATRIGEL basement membrane matrix. Neutrophils were lysed to measure total VEGF release, and VEGF exp ression was detected using Western blot analysis. Results: Lipopolysaccharide and TNF-alpha stimulation resulted in significa ntly increased release of PMN VEGF (532 +/- 49 and 484 +/- 80 pg/mL, respec tively; for all, presented as mean +/- SEM) compared with control experimen ts (32 +/- 4 pg/mL). Interleukin 6 and Fas had no effect. Culture supernata nts from LPS- and TNF-alpha-stimulated PMNs also resulted in significant in creases (P<.005) in macrovascular and microvascular endothelial cell prolif eration and tubule formation. Adding anti-human VEGF-neutralizing polyclona l antibody to stimulated PMN supernatant inhibited these effects. Total VEG F release following cell lysis and Western blot analysis suggests that the VEGF is released from an intracellular store. Conclusion: Activated human PMNs are directly angiogenic by releasing VEGF, and this has important implications for inflammation, capillary leak syndr ome, wound healing, and tumor growth.