Abstract
Purpose::
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.
Methods::
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.
Results::
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.
Conclusions::
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.