Abstract
Purpose :
Exfoliation Glaucoma (XFG) is associated with high intraocular pressure (IOP) and is difficult to treat. XFG is manifested by the presence of amyloid-like deposits of exfoliation material (XFM) on anterior segment tissues, most prominently on the lens surface at the pupillary border. Our goal was to develop a relevant system to study and induce formation of XFM by gene transfer of its components; to focus on lens capsule organotypic cultures (LC) from capsulorhexis specimens; and to characterize differences between normal and XFG patients.
Methods :
Numerous culturing conditions were evaluated to optimize LC maintenance plus growth of lens epithelial (LE) cells up to 4 weeks. Overproduction of XFM components clusterin (CLU) and LOXL1 was achieved by infecting with DDK-tagged Ad.CLU and Ad.LOXL1. LCs were exposed to conditioned media (CM) from human iris pigment epithelial (hIPE) cells. IHC was performed with CLU, LOXL1, DDK, elastin (ELN), fibrillin I (FBN1) & collagen IV antibodies under permeabilized and non-permeabilized conditions. Images were acquired with Olympus XL7I and FV3000RS confocal/FV-OSR super-resolution microscopes and analyzed with CellSens software.
Results :
Specimens adhered best to uncoated cover-glass multi-chambers 12-24 h post-surgery in 20% FBS/IMEM medium. LE cells start growing from the LC at 1 wk and continue for 4 wks. In LCs from normal patients, IHC shows abundant LOXL1 production and a robust FBN1 network which can form bundles. LOXL1 overexpression induced formation of a FBN1 tangled plaque, and exposure to CM from hIPE cells overexpressing LOXL1 had a marked effect on LE cell morphology. LCs of XFG patients exhibit abundant abnormal aggregated deposits of FBN1, CLU and ELN, not seen in normal LCs. Confocal optical sections from Z-stacks showed LE cell movement from beneath to the top of the capsule. Further, XFS outgrowing cells produce more and differently shaped ELN deposits than normal controls.
Conclusions :
We show that four key XFM proteins are produced by the LE layer beneath the capsule and that the XFG LC cultures show cell/ECM abnormalities when compared with normal controls. Because of the high availability of capsulorhexis specimens, these organotypic cultures could be of great value to study mechanisms involved in the formation of XFM in XFG.
This is a 2020 ARVO Annual Meeting abstract.