Purchase this article with an account.
Qitao Zhang, Feriel Presswalla, Carol Charniga, Jeffrey Stern, Sally Temple, David N Zacks, Debra A Thompson, Jason Miller; A primary retinal pigment epithelial culture model for evaluating therapeutic targets in dry macular degeneration. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5015.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
In a primary culture system, we seek to simultaneously reproduce the two key features of aged, AMD-susceptible retinal pigment epithelium (RPE) - the intracellular accumulation of indigestible, autofluorescent material (IAM) and the extracellular accumulation of lipid-rich debris. Using this model, we are probing a range of degradative and lipid homeostatic pathways for therapeutic potential.
Polarized human fetal and human adult RPE cultures (P1) were subjected to at least 14 consecutive days of a high concentration mixture of UV-treated and untreated bovine outer segments (OS) delivered with the bridging ligands human Protein S and MFG-E8. Accumulation of IAM was evaluated by spectral analysis on a confocal microscope. Dynamics of intracellular lipid accumulation were tracked with lipid droplet staining. Accumulation of extracellular lipid was monitored by APOE and cholesterol immunostaining of the basolateral-facing Transwell membrane and Western blots of the apical and basolateral media.
In cells fed OS, IAM accumulated significantly faster in the presence of Protein S and MFG-E8. The autofluorescence spectrum of IAM from cells fed UV-treated OS vs. those fed untreated OS were identical, and both were slightly blue-shifted from the spectra of endogenous lipofuscin. Both UV-treated and untreated OS contained retinoids, but UV treatment induced increased autofluorescence in both OS-protein and -lipid fractions. Adult RPE cultures accumulated markedly more IAM than fetal cultures, providing a platform for exploring factors impacting OS degradation. Over 30 small molecules and proteins were tested for their ability to upregulate the degradative autophagy pathway in RPE, and positive hits are being tested for their ability to reduce the accumulation of IAM. We are also investigating the effects of IAM on the dynamics of lipid droplet formation and dissipation, as well as cholesterol and APOE buildup. Small molecule modulators of RPE lipid synthesis and degradation are being tested for their ability to alter lipid droplet, cholesterol, and APOE dynamics.
Our comparative human vs. fetal primary culture model of dry AMD provides a platform for screening pathways, small molecules, and proteins that can alter the accumulation of lipofuscin and drusen, two hallmarks of aged, AMD-susceptible RPE.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
This PDF is available to Subscribers Only