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C. M. McVicar, A. Rice-McCaldin, T. Curtis, A. W. Stitt, T. A. Gardiner; Angiogenesis Induced by EGF Is Mediated by Autocrine VEGF. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1379.
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Epidermal growth factor (EGF) plays a major role in retinal development and is expressed by several types of neurones in adulthood. EGF is pro-angiogenic in many situations but has not been studied in retinal angiogenesis (AG). As normal neuro-vascular relationships are essential for vascular cell survival we studied the role of EGF in angiogenesis by retinal vascular endothelial cells (RVEC) in a 3-D duplex Matrigel culture system. EGF-induced angiogenic signalling was studied with several agonists and antagonists and with a neutralising antibody to VEGF.
RVEC from bovine retina were plated in 30µl spots of 50% Matrigel and formed endothelial tubular networks by 24 hours. Test substances were added to a second layer of Matrigel and superimposed on the primary culture spots. Angiogenic sprouting from the primary to secondary gel layers were counted after a further 24 hours and the mean calculated in 10 spots per treatment group.
EGF at 20ng/ml increased the number of angiogenic sprouts obtained with control Matrigel by between +60% and +150%. As the nitric oxide synthase inhibitor L-NAME reduced EGF-induced AG to control values, the downstream signalling from eNOS was investigated with ODQ, an inhibitor of soluble guanylate synthase (sGC), the stable cGMP analogue 8-bromo-cGMP, and KT5823 an inhibitor of cGMP-dependent protein kinase (PKG). The sGC and PKG inhibitors reduced AG to sub-control values and 8-bromo-cGMP increased it to levels comparable to EGF (+80%). Previous studies implicated PKG activation of Raf-1 in VEGF-mediated angiogenic signalling. We therefore inhibited the Ras-Raf-MEK-ERK pathway with PD98059 and Apigenin, each of which reduced AG to sub-control levels. To test whether EGF-induced AG was mediated through autocrine VEGF, EGF and VEGF-induced cultures were treated with an anti-VEGF neutralising antibody (R&D systems) or irrelevant isotype-specific IgG. EGF, VEGF and each growth factor with irrelevant IgG increased AG to approx+100% of control Matrigel (p<0.0001), while anti-VEGF reduced both EGF-induced and VEGF-induced AG to control levels.
EGF appears to exert its angiogenic on retinal vascular endothelial cells by stimulating the autocrine secretion of VEGF. EGF secretion by retinal neurones can exert pro-angiogenic effects via stimulation of endothelium-derived VEGF.
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