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Maria Notara, Gabriele Braun, Marie Luise Dreisow, Felix Bock, Claus Cursiefen; Avastin effects on human limbal epithelial cell function and phenotype in vitro. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4343.
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© ARVO (1962-2015); The Authors (2016-present)
Topical application of the VEGFA inhibitor bevacizumab (Avastin) is used for antiangiogenic therapy at the cornea. While clinical studies have suggested that this off-label use of Avastin is well-tolerated with relatively few adverse effects, the impact of the drug on limbal epithelial stem cells is unknown. In this study, the effect of Avastin on the functionality and phenotype of limbal epithelial cells was investigated
The effect of different concentrations of Avastin (0 mg/ml, 0.125mg/ml, 0.5mg/ml and 1mg/ml) on human limbal epithelial cells was investigated in terms of proliferation using an alamar blue assay and migration by a scratch wound assay (SWA). Changes in phenotype were assessed using a colony forming efficiency (CFE) assay as well as immunostaining and QPCR of markers associated with basal limbal epithelial cells, differentiated corneal epithelial cells and putative stem cells including integrinβ1, cytokeratin 3 and ΔΝΡ63∝ respectively. The effect of the treatment in RNA expression of VEGFA, C and D and their receptors VEGFR1, 2 and 3 was also assessed.
The different treatment groups featured no difference in proliferation and CFE as well as morphology and marker expression by immunostaining. A small but significant delay in wound healing of all the Avastin treated groups was detected at an early timepoint of 2h. QPCR assessment indicated a small but significant increase of integrinβ1 and a strong expression in cytokeratin 3 with the increasing Avastin concentrations while ΔΝΡ63∝ RNA levels remained unaffected. VEGFA RNA expression also significantly increased while VEGFC and D were unchanged. No differences were observed in the expression of VEGFR1, 2 and 3.
This study exhibited previously unknown short-term effects of Bevazizumab in cultured human limbal epithelial cells: Avastin-induced a dose-responsive increase of cytokeratin 3 expression, indicating a shift towards a more differentiated phenotype, as well as a delay in wound closure correlating with the delayed wound healing observed clinically with the use of Avastin. The increase in VEGFA RNA levels may reflect an autocrine defence mechanism of the limbal epithelial cells as they attempt to compensate for the neutralisation effect of Avastin. The data obtained through this in vitro approach confirm the effects of Avastin while elucidating phenotypical and functional changes in limbal epithelial cells.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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