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Javier Adrián Calles, Mario Crespo-Moral, Antonio Lopez-Garcia, Yolanda Diebold, Ocular Surface Group; An in vitro model using corneal cells to test antiangiogenic activity. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4506.
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© ARVO (1962-2015); The Authors (2016-present)
Vascular endothelial growth factor (VEGF) is widely viewed as the main proangiogenic stimulus in pathological neovascularization of eye tissues including the cornea. There is a growing interest in the discovery of new molecules that inhibit VEGF and only a few in vitro corneal models of neovascularization. This work aims at designing an in vitro model to study antiangiogenic activity of drugs based on VEGF production by corneal cells.
Human corneal epithelial (HCE) cells were grown in DMEM/F12 medium with or without serum as basal conditions. Then, cells were exposed to IL-6 (50 or 100 ng/ml) for 24 or 48 h. VEGF release was measured in cell culture supernatants by ELISA after 24, 48 and 72 h. In another set of experiments, cells were scratched with a pippete tip to simulate a wound and maintained in serum-free medium for 24 h. Immediatly after the scratch, either same IL-6 concentrations or none were added, and release of VEGF by HCE cells during wound closure was measured. Conjunctival epithelium and retinal pigment epithelium cell lines were used as controls for unstimulated secretion of VEGF. Data are expressed as mean ± SEM, and were analyzed by the Student t test.
HCE cells released detectable VEGF levels that were about 6 times lower than those produced by control cell lines. Basal release of VEGF by the three cell lines occurred in a time-dependent manner that was significantly affected by the presence of serum in culture medium. 50 ng/ml but not 100 ng/ml IL-6 significantly increased VEGF expression in HCE cells, being 85% and 66% higher for 24 and 48 h respectively (p < 0.05). A 20% reduction in VEGF release was observed in scratched cells (p < 0.05); however, the presence of IL-6 returned VEGF levels to those of the control uninjured cells.
Corneal cells responded to serum or IL-6 stimulation by increasing VEGF secretion. These cells responded with a reduction in VEGF secretion when they were wounded, and IL-6 partially compensated that reduction. These findings indicate that corneal cells are reactive in vitro to stimuli affecting VEGF production. This model could be useful to test antiangiogenic drugs for corneal neovascularization.
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