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A.B. El-Remessy, N. Tsai, R.B. Caldwell; Is Superoxide Anion a Mediator of Vascular Endothelial Growth Factor (VEGF) Angiogenic Function in Retinal Endothelial Cells? . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2913.
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Purpose: Recent evidence has shown that nitric oxide (NO) plays an important role in the mitogenic signaling pathway of vascular endothelial growth factor (VEGF). Peroxynitrite is a highly reactive oxidant formed by the rapid combination of NO with superoxide anion. The purpose of this study was to investigate the involvement of superoxide anion and peroxynitrite in VEGF’s angiogenic function. Methods: Dihydroethidine imaging techniques were used to determine the effects of VEGF on the formation of superoxide anion in cultured bovine retinal endothelial (BRE) cells. A tube formation assay with three-dimensional type I collagen gels was used to test whether or not superoxide anion is required for VEGF’s action in stimulating BRE cell differentiation into capillary-like structures. An assay for tyrosine nitration was used to investigate whether or not VEGF’s action in promoting superoxide anion formation results in the formation of peroxynitrite. Results: VEGF (60 ng/ml) caused significant increases in superoxide anion formation in BRE cells. This effect was totally blocked by SOD (100 U/ml) and significantly inhibited by the specific NADPH oxidase inhibitor apocynin (15 µM). Superoxide formation was not reduced by the NO synthase inhibitor L-NAME (0.5 mM) or by treatment with supplemental arginine (1 mM), indicating that NO synthase is not a source of superoxide. The increase in superoxide formation was followed by a significant increase in nitrotyrosine formation that was inhibited by SOD, apocynin and L-NAME, but was unaffected by catalase (100 U/ml). The results of the tube formation assay showed that VEGF caused significant increases in BRE cell elongation and alignment into capillary like structures which were blocked by apocynin and SOD. Conclusions: Taken together, these data suggest that superoxide anion plays an important role in the angiogenic signaling pathway stimulated by VEGF and that NADPH oxidase is a likely site of superoxide anion formation. Moreover, the combination of superoxide with NO to form peroxynitrite may also have a key role in VEGF’s angiogenic function. Treatments targeted at NADPH oxidase or peroxynitrite may be effective in anti-angiogenic therapy.
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