Abstract
Purpose :
Age-related macular degeneration (AMD) affected approximately 2 million Americans in 2010, and this number is expected to more than double by 2050 (National Eye Institute). In diseases like AMD, abnormal angiogenesis can lead to vision loss or blindness. While the cause of angiogenesis is unknown, previous work suggests that retinal pigment epithelial (RPE) cell detachment contributes through the imbalanced secretion of angiogenic proteins. Increased expression of pro-angiogenic proteins, like vascular endothelial growth factor (VEGF), and decreased expression of anti-angiogenic proteins, such as pigment epithelium-derived factor (PEDF), both contribute to angiogenesis. To see how RPE cell detachment affects angiogenic protein expression, we used micropatterning methods to control cell growth patterns. This method created affordable stencils that can be tuned to various stages of RPE degeneration and control cell detachment in vitro.
Methods :
Real fundus images were analyzed with ImageJ software to isolate areas of retinal degeneration. These areas were combined using AutoCAD into a single shape for easier manipulation in culture while maintaining the total area of detachment. Soft lithography, a technique to fabricate shapes using a flexible mold, was used to create polydimethylsiloxane (PDMS) stencils. These stencils were placed in culture plates and seeded with primary porcine RPE cells that grew until confluency. The stencils were then removed, which introduced an area void of cell growth. Enzyme-linked immunosorbent assays and immunocytochemical staining will be used to determine how angiogenic protein expression changes with RPE cell detachment.
Results :
Patterns with an area of approximately 47.6 mm2 were created in AutoCAD. These patterns were formed into stencils that represented areas of RPE cell detachment and effectively controlled the growth of primary porcine RPE cells. One day after stencil removal, RPE cells began growing in the open spaces and angiogenic protein secretion of the RPE cells changed.
Conclusions :
Engineered models of RPE cell detachment can be created from analyzed fundus images. The results of this research will elucidate relationships between varying stages of detachment and angiogenic protein expression. This understanding may increase the efficacy of current treatments and lead to additional therapeutics for retinal pathologies.
This is a 2021 ARVO Annual Meeting abstract.