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Amandio A Rocha-Sousa, Rita Silva-Gomes, Gloria Conceicao, Ana Filipa Moleiro, Cristiana Santos, Sara Azevedo-Pinto, Paulo Pereira-Silva; Ghrelin’s effects in choroid retinal cell proliferation, apoptosis and migration under a hyperglycemic environment. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4300.
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© ARVO (1962-2015); The Authors (2016-present)
Ghrelin is a peptide present in many organs and tissues. New roles have been discovered, including anti-apoptotic, anti-angiogenic and proliferative effects on several cell lines. Recently, ghrelin has been implicated in the pathophysiology of proliferative retinopathy, although its true involvement remains unclear. We tested the hypothesis that ghrelin could alter the proliferation, apoptosis and migration of primate choroid retinal endothelial cells, cultured under hyperglycemic conditions.
RF/6A cells were incubated for 24h with different concentrations of glucose (0-250mM). Colorimetric immunoassay was used for the quantification of cell proliferation, based on the measurement of BrdU incorporation during DNA synthesis. Cell apoptosis was assessed by TdT-mediated dUTP-X nick end labeling (TUNEL) technique and cell migration was assessed using the wound-healing assay. For each assay, we investigated ghrelin's effect by adding it to the cell culture for 24h, with increasing concentrations: 10-10,10-9,10-8,10-7,10-6 and 10-5nM. We compared cell proliferation, apoptosis and migration in different glucose concentrations (0, 10,100 and 250mM) with and without adding ghrelin to the medium. Concerning proliferation and migration assays, positive controls had VEGF added to the medium. DNAse was used on TUNEL assay in the positive control group. One way ANOVA was used for statistical analysis.<br />
Glucose seems to potentiate cell proliferation at concentrations from 0 to 100 mM, while at higher concentrations (150-300 mM) it shows an inhibitory tendency. On the other hand, increasing concentrations of glucose show a reduction on the migration distance. Concerning the apoptosis assay, no significant differences in the number of apoptotic cells was found between the different glucose concentrations. At 10 and 100 mM of glucose, ghrelin seems to inhibit cell proliferation at 10-7 and 10-6 nM (p<0,05). About the migration assay, at the concentration of 10-8 nM, ghrelin decreases cell migration in all glucose concentrations tested (p<0,05).<br />
Ghrelin reduces RF/6a cells proliferation and migration cultured under different glucose concentrations but seems to have no effect on apoptosis.<br />
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