Abstract
Purpose :
RPE cell attachment to extracellular matrix (ECM) proteins is critical to retinal health, however a number of extracellular and endogenous factors may perturb or enhance cell attachment, including modifications to ECM proteins and pigmentation. This study uses in vitro modeling to examine the role of the cytokine, interleukin-6 (IL-6) in RPE cell attachment to ECM proteins.
Methods :
ARPE-19 derived ECMs were used. Matrices were glycated with either glycolaldehyde (GA) or methylglyoxal (MG). Control ECMs were treated with PBS. ARPE-19 cells were plated onto ECM coated plates or tissue culture treated (TCT) plates and harvested either 30 minutes or 24 hours after seeding. IL-6 expression was determined using qPCR and expression was evaluated relative to cells plated onto TCT plates. Rate of cell attachment was evaluated over the course of one hour after seeding by hemocytometer counting of unattached cells. Mass spectrometry was used to identify modifications to cell-matrix attachment sites. Statistical analysis was performed using one-way or two-way ANOVA.
Results :
30 minutes after seeding, IL-6 expression was significantly induced in ARPE-19 cells seeded onto an unmodified matrix compared to cells seeded onto TCT plates (p ≤ 0.01) or glycated matrices (p ≤ 0.05). Cells seeded onto an unmodified matrix showed a 3269±1597% increase in IL-6 expression, while cells seeded onto GA or MG modified matrices showed a 113±15% or 116±20% increase, respectively. 24 hours after seeding, a significant decrease in IL-6 expression was observed in cells seeded onto unmodified or glycated matrices compared to cells seeded onto TCT plates (p ≤ 0.01, p ≤ 0.001). IL-6 expression in cells seeded onto an unmodified matrix decreased to 12±5% and to 4±1% and 8±2% in cells seeded onto GA or MG treated matrices. Over the course of an hour, a significant decrease in cell attachment was observed in cells seeded onto a glycated matrix versus cells seeded onto an unmodified matrix (p ≤ 0.0001).
Conclusions :
IL-6 is significantly induced in RPE cells attaching to unmodified ECM proteins. Glycation of ECM proteins hinders the rate of cell attachment to these matrices. This evidence suggests that IL-6 may be a necessary factor in RPE cell attachment to ECM proteins. It is possible that endogenous factors such as pigmentation may also play a role in RPE cell adhesion and IL-6 expression and secretion may serve as a marker for cell attachment.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.