Abstract
Purpose :
XFS in the eye causes the formation of deposits on anterior segment tissues that can lead to the development of exfoliation glaucoma. Clinically, deposits are seen on the lens surface. Our goal was to define human capsulorhexis cultures as an XFS model by determining if lens exfoliation material (XFM) was produced by lens epithelial cells (LE) or deposited externally. To analyze local distribution of XFM components LOXL1, ELN and FBN1 in XFS & normal capsulorhexis tissues and to investigate LOXL1’s role in the formation of XFS aggregates.
Methods :
Lens-capsules (LC) were harvested in tissue culture (TC) media during capsulorhexis surgery (IRB approved). Four h post-surgery, specimens were stained with trypan blue, transferred to a 2-chamber dish with coverglass bottom (4.2 cm2), washed, and incubated floating in media overnight. Curled LCs were flattened with craft brushes (LE layer down), incubated with media drops 2 days for attachment, and fed normally for 2-4 weeks. IHC was performed in the same dish with LOXL1, ELN, FBN1, Collagen IV and DDK antibodies. Images were acquired through the chamber coverglass (unmounted) by fluorescence microscopy and analyzed with CellSens. LOXL1 overexpression was achieved by infection with DDK-tagged Ad.LOXL1 (variant G-G).
Results :
83 LCs were processed. LOXL1, ELN, and FBN1 are abundantly secreted by LEs and can raise to the surface through the 14 nm capsule membrane. LCs from XFS patients show patches of aggregates which stain heavily with LOXL1, ELN and FBN1. The XFS cells’ morphology exhibit an extracellular ring reminiscence of a pericellular matrix. Interestingly, a similar extracellular ring is observed in LEs transduced with Ad.LOXL1. ELN shows a globular conformation in the aggregate and a fiber shape away from it. Overexpression/secretion of LOXL1 induces LOXL1/FBN1 and LOXL1/ELN deposit-like aggregates. FBN1 forms microfibril bundles in most transduced LCs.
Conclusions :
Lens organotypic cultures from capsulorhexis of normal- and XFS- cataractous lenses represent a unique model to identify mechanistic differences between normal and XFS cells. Morphological and functional similarities between XFS- and LOXL1-transduced cells reaffirm the functional relevance of LOXL1 and the prospect of using this gene to manage the disease. This model could also be used to search for potential treatments to disassociate and/or prevent XFM aggregates.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.