Direct measurement of VEGF-induced nitric oxide production by choroidal endothelial cells

Vascular endothelial growth factor (VEGF) and nitric oxide (NO) seem to be involved in the process of angiogenesis, but their interactions are not clearly understood. The aim of this study was to investigate the influence of VEGF on NO production of choroidal endothelial cells (CEC) and its importance in angiogenesis. Experiments were performed using cultured bovine CEC. Basal NO release of unstimulated CEC was measured and compared to NO release of VEGF-stimulated CEC (1, 10, and 100 ng/ml). Further, cells were pretreated with N(omega)-nitro-L-arginine methyl ester (L-NAME, 1 and 2 mM) and incubated with and without VEGF (10 ng/ml) to investigate the effect of blocking NO synthase. NO release into the medium was assessed by an amperometric NO sensor. To show the importance of NO in angiogenesis, proliferation and migration of CEC were measured after VEGF stimulation and in the presence or absence of L-NAME (1 and 2 mM). Unstimulated CEC continuously produced low levels of NO. Stimulation of the cells with VEGF resulted in a dose-dependent increase in NO release. The time course after stimulation with 10 ng/ml VEGF was characterized by a prompt initial rise up to 140% of unstimulated levels and a subsequent sustained increase over 120 min. Pretreatment with L-NAME attenuated the VEGF-induced response. L-NAME incubation alone led to a reduction in basal NO release. L-NAME also significantly diminished the VEGF-enhanced CEC proliferation and migration. The results demonstrate that VEGF enhances the formation of NO in cultured CEC. The blockade of NO production reduces CEC proliferation and migration, an effect which may be important for controlling angiogenesis, especially in reducing neovascularization in age-related macular degeneration in the eye.