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TA Gardiner, AM McCaldin; Exogenous Nitric Oxide Inhibits Growth Factor-Induced Angiogenesis by Retinal Microvascular Endothelial Cells In Vitro . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2745.
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Purpose:Sennlaub et al (J Clin Invest. 2001;107:717-725) have shown that intra-retinal angiogenesis is potentiated and extra-retinal neovascularization inhibited during the proliferative phase of oxygen induced retinopathy (OIR) in mice lacking inducible nitric oxide synthase (NOS-2). Because of the multifarious effects of nitric oxide in the complex tissue environment of the retina this study was performed to assess whether nitric oxide per se is inhibitory to angiogenic invasion by retinal microvascular endothelial cells (RVEC) in a simple in vitro model. Methods:Bovine RVEC were suspended in Martigel and 40ml aliquots spotted on 3cm plastic petri dishes and allowed to gel. Overnight incubation (approx 18 hours) was sufficient for the development of vascular networks within the gels. At this time the medium was drained and a further coating of liquid Matrigel containing test substances was layered over the original gels and solidified as above. The duplex gels were then covered with growth medium and incubated for 24, 48 and 72 hours. On phase microscopy a distinct phase-dark line clearly demarcated the interface of the original gel cultures and the outer secondary layer containing the test materials. The number of angiogenic sprouts invading the secondary layer could then be counted. To assess the effect of exogenous nitric oxide (NO) on in vitro angiogenesis invasion, the NO generators sodium nitroprusside (SNP) and s-nitroso-n-acetyl-penicillamine (SNAP), each at 2mM, were added to the outer gel, with or without epidermal growth factor (100ng/ml) as chemo-attractant. In control groups the outer layer contained Matrigel alone or Martigel+EGF. Sets of 10 gel cultures were counted for all test and control groups and the experiment was performed 3 times, each on a separate cell isolate. Statistical comparisons were made using a one-way ANOVA and a Tukey-Kramer test for multiple comparisons. Results:EGF alone elicited the greatest angiogenic response while EGF-stimulated angiogenesis was significantly inhibited by the presence of the NO generators SNP and SNAP. However, it was of interest that in the absence of EGF stimulation, both SNP and SNAP consistently proved to be more angiogenic than control Matrigel. Conclusion:The NO generators SNP and SNAP stimulate in vitro angiogenesis but inhibit angiogenesis induced by EGF. These results suggest a direct inhibitory effect of exogenous NO on intra-retinal angiogenesis during OIR and add to the explanation of the observed potentiation of the response in NOS-2 knockout mice.
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