Abstract
Purpose :
Retinal pigmented epithelium (RPE) cells are located between the choroid and photoreceptors within the eye and are essential to provide nutrients from blood to rods and cones, as well retinoids of the visual cycle. Vision loss and ocular diseases are attributable to the degeneration or dysfunction of the RPE cells, leading to blindness. One of the major ocular problem from RPE dysfunction is macular degeneration. Age-related macular degeneration (AMD) can be frequently diagnosed in elderly patients. The purpose of this study is to provide important information on the molecular pathway on RPE survival to minimize rejection of RPE cell in transplantation to restore vision in AMD patients. This study poses as a cell model for iPSC-RPE in either 5.5 or 30mM glucose conditions in proinflammatory or hypoxic environments to determine if IL8 can act as possible cell protectants against different glucose concentrations in proinflammatory and hypoxic conditions.
Methods :
iPS-RPE were seeded in fetal RPE media of MEM, N1 supplement, glutamine, nonessential amino acids, taurine .25mg/mL, hydrocortisone 10ng/mL, triiodothyronine 13ng/mL,15%FBS, 1%pen/strep, in a humidified 5%CO2 incubator with a temperature of 37°C. In order to determine the iPSC-RPE cell proliferation/viability, cell viability prior to treatment, 12hrs after treatment and 72hrs after treatment was determined using the Trypan Blue Method. Cells were treated with LPS 10µg/mL, Cobalt Chloride 1mM/mL, IL8 12.5ng/mL or 25ng/mL, 0mM/mL, 5.5mM/mL, or 30mM/mL of glucose. Cell culture media supernatants was collected and stored in -80°C. MCP-1 and RANTES were identified using a multiplex ELISA.
Results :
Wells treated with 12.5ng/mL of IL8 in 30mM of glucose went from 11,700 cells at 12hrs to 2,060,000 cells at 72hrs. In comparison, wells treated with 25ng/mL of IL8 and increased from 11,700 cells at 12hrs to 258,000 at the end of treatment at 72hrs. RANTES showed a significant increase at 72hrs whereas MCP-1 expressed significant increase at 12hrs. The results from a Two Way ANOVA calculated the interaction effect and identified RANTES expression was significant at 72hrs comparable to MCP-1 expression at 12hrs.
Conclusions :
Increased viable cell number in response to IL8 treatment suggest IL8 may provide protection for iPSC-RPE and MCP-1/RANTES expression. This study was essential in understanding possible therapeutic treatment models for AMD.
This is a 2021 ARVO Annual Meeting abstract.