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A.M. R-McCaldin, T.M. Curtis, T.A. Gardiner; Endogenous Nitric Oxide Mediates Egf-induced Angiogenesis by Retinal Vascular Endothelial Cells In Vitro . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2916.
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Purpose: We have previously shown that exogenous nitric oxide (NO) and epidermal growth factor (EGF) are pro-angiogenic when applied to preformed networks of endothelial tubes derived from retinal vascular endothelial cells (RVECs). The present study begins to examine the signalling mechanisms involved in nitric oxide and EGF-mediated modulation of in vitro retinal angiogenesis focusing specifically on the role of soluble guanylate cyclase and endothelial nitric oxide synthase. Methods: To study retinal angiogenesis in vitro we employed a novel duplex culture in which retinal vascular endothelial cells (RVECs) were allowed to form vascular networks in a primary 3-D Matrigel culture unto which a secondary gel layer containing test substances was superimposed. Bovine RVEC were suspended in Matrigel and 40ul 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 a further 24 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. Groups of 10 individual cultures were treated with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase and L-NAME an inhibitor of nitric oxide synthase in the presence and absence of epidermal growth factor (EGF) or the nitric oxide donor sodium nitroprusside (SNP). Each experiment was performed in triplicate and statistical analysis was carried out using one way ANOVA with a Tukey-Kramer post test for multiple comparisons. Results: The guanylate cyclase inhibitor ODQ significantly reduced angiogenesis induced by the nitric oxide donor SNP (p<0.0001). However, ODQ also inhibited EGF-induced angiogenesis (p<0.0001) as did the nitric oxide synthase inhibitor L-NAME (p<0.001). Conclusions: Angiogenesis by retinal vascular endothelial cells may be induced by nitric oxide from endogenous or exogenous sources. EGF-induced angiogenesis is mediated by endogenous nitric oxide synthase via NO-stimulation of soluble guanylate cyclase.
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