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Juan Sebastian Pena, Maribel Vazquez; Characterization of VEGF-EGFR interaction in Müller glia to evaluate migration within controlled microenvironments. Invest. Ophthalmol. Vis. Sci. 2020;61(7):695.
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© ARVO (1962-2015); The Authors (2016-present)
Müller glia (MG) are central to the response upon retinal insult, becoming recent therapeutic targets for diverse retinopathies. In response to retinal injury, MG upregulate production of pro-angiogenic factors, such as vascular endothelial growth factor (VEGF) to support the elevated metabolic activities of damaged retina. Furthermore, VEGF stimulates de/differentiation of neural stem-like cells, while upregulation of Epidermal Growth Factor Receptor (EGF-R) facilitates migratory responses during tissue repair of MG. This project examines the relationship between VEGF signaling and EGF-R expression leading to MG chemotaxis within a microfluidic device, previously developed by our group on the scale of adult retina.
Gene expression levels of EGF-R were measured using qPCR for MG treated with either EGF or VEGF. MG were plated on borosilicate glass chambers, and EGFR expression was evaluated through immunocytochemistry methods. Lastly, Cell migration in response to EGF or VEGF stimulus was examined in our microfluidic device using four testing conditions: (1) MG stimulated by EGF; (2) Stimulation by VEGF; (3) Stimulation by VEGF for one hour, then exposed to EGF fields; and (4) Control. Computational modeling of the growth factor gradients was also modeled within our microfluidic device.
EGF-R expression was dramatically increased by 18.9-fold in MG stimulated with VEGF, conversely to 2.2 fold when stimulated with EGF, both normalized with respect to control. Intensity levels for the three groups were measured in response to EGF signaling, where VEGF stimulation led to upregulation of EGFR expression. Path length of motile MG was compared against control conditions in the device. MG displayed higher chemotaxis when exposed to the mixed condition (VEGF/EGF) than when exposed to EGF or VEGF alone. Statistical significance was measured (p<0.01).
Data illustrate that VEGF stimulated upregulation of EGFR more than its cognate ligand, suggesting crosstalk between VEGF and EGF-R signaling pathways to regulate movement. Our study used a quantitative platform to show the interplay between VEGF and EGF-R as occurs during retinopathies, following by chemotactic responses of MG.
This is a 2020 ARVO Annual Meeting abstract.
VEGF/EGFR interaction: A) VEGF-EGFR expression. B) Quantification of intensity. C) Microfluidic device used. D) Migration analysis.
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