Abstract
Purpose :
According to the National Eye Institute, vision impairment caused by diseases such as age-related macular degeneration (AMD) and diabetic retinopathy affected approximately 10 million Americans in 2010, and this number is expected to double by 2050. Abnormal angiogenesis can often lead to blindness in these retinal diseases. While the exact cause of abnormal retinal angiogenesis is unknown, retinal pigment epithelial (RPE) cells experiencing detachment likely play a significant role by overexpressing pro-angiogenic proteins, such as vascular endothelial growth factor (VEGF), and decreasing expression of anti-angiogenic proteins, like platelet epithelial derived growth factor (PEDF). To understand how RPE cell-cell detachment promotes angiogenesis, we used micropatterning: a novel method of creating patterned surfaces to control the spatial arrangement of cells.
Methods :
To develop the micropatterned surfaces, fundus images of various stages and geometries of retinal degeneration were analyzed using ImageJ software to calculate their perimeter and area. AutoCAD was used to create patterns based on the fundus images, which were then formed into polydimethylsiloxane (PDMS) stencils using soft lithography. The PDMS stencils will be compared to the original CAD drawings to determine the efficacy of the micropatterning methods. When applied to a monolayer of RPE cells, these stencils control cell growth and detachment. The change in angiogenic proteins will be evaluated with an enzyme linked immunosorbent assay (ELISA). VEGF and PEDF expression will be visually characterized using immunocytochemistry (ICC) staining. Results from both assays will be used to correlate stages of retinal degeneration with angiogenesis.
Results :
Varying levels of RPE degeneration were replicated in vitro through fundus image analysis and micropatterning. The ELISA results indicate which specific angiogenic factors are being produced and the change in concentration of these factors as a result of RPE cell-cell detachment. ICC will enable us to visually represent VEGF and PEDF expression and how they correlate with cell-cell detachment.
Conclusions :
Engineering techniques can be used to create tunable models of RPE cell-cell detachment and degeneration. The results of this research will elucidate the production of specific angiogenic compounds and how different levels of degeneration can contribute to angiogenesis.
This is a 2020 ARVO Annual Meeting abstract.