Abstract
Purpose :
With the aging population, novel treatments are needed to treat and slow the progression of age-related macular degeneration (AMD). A prominent feature of AMD is the accumulation and secretion of fatty deposits, or drusen, by retinal pigment epithelial (RPE) cells in the posterior segment of the eye. The purpose of this work was to construct a high-throughput in vitro screening platform to access compounds that modulate lipid and cholesterol production in primary human RPE cells. We demonstrate that starvation-induced lipid and cholesterol biosynthesis in RPE cells can be modulated through constructed small molecule compounds.
Methods :
Human primary, adult RPE cells were isolated and differentiated in vitro to confluency. Once confluent, differentiated RPE cells were cultured in 10% serum, 1% serum, or no serum for a total of 7 days. Compounds or the vehicle control were added at day 4 to evaluate effects on RPE lipid and cholesterol metabolism and cell stress. After 7 days in culture, RPE cells were immunostained and high-content imaged for Filipin III, Oil Red O, LysoTracker®, and CellROX®, to assess production of esterified and unesterified cholesterol, neutral lipids, lysosomes, and reactive oxygen species. Genes involved in lipid and cholesterol metabolism were also analyzed through qPCR. Conditioned media from serum-starved RPE cells was analyzed in a Luminex assay to determine secreted AMD biomarkers.
Results :
A significant increase in Filpin III, Oil Red O, LysoTracker®, and CellROX® expression was detected in RPE cells cultured at low serum and no serum when compared to RPE cells cultured in high serum. Low serum and serum starvation also induced significant expression of the genes SCD, HMGCR, ACAC1, PPARG, and CEBPA. Furthermore, biomarkers of AMD, complement component 2 and MMP-2, was increased in the conditioned media of serum-starved RPE cells. After the introduction of small molecule compounds, we demonstrate that lysosomes, cell stress, and lipid and cholesterol metabolism was significantly upregulated or downregulated depending on the analog of the created small molecule compound.
Conclusions :
This platform provides an effective means to induce cholesterol and lipid production in human primary RPE cells, and allows for the analysis of therapeutically relevant endpoints for AMD in drug screening applications.
This is a 2020 ARVO Annual Meeting abstract.