Abstract
Purpose :
Pseudoexfoliation (PEX) syndrome is a multifactorial complex disorder of the extracellular matrix with important clinical ramifications including a particularly severe and common type of glaucoma. Progress in understanding of PEX pathogenesis and development of specific treatments is hampered in good part by lack of suitable experimental models. Here, we developed a cell culture model using PEX-relevant intraocular cells with appropriate genetic background, which recapitulates key features of the disease including PEX material formation and accumulation.
Methods :
Primary human iris stroma cells were isolated from 1x1mm iridectomy specimens obtained from patients with PEX glaucoma during routine trabeculectomy after informed consent (n=20). Specimens from POAG patients served as control. Stromal fibroblasts were enriched from mixed cell cultures by standardized protocols. Extracellular matrix formation and deposition was stimulated in 2D and 3D-spheroid cultures by TGF-ß1 and macromolecular crowding with Ficoll 400. Cell cultures were analyzed by immunocytochemistry, transmission electron microscopy and qPCR.
Results :
Stromal fibroblasts were cultured for 5 passages and characterized by expression of vimentin, fibronectin, PDGFRA, S100A4, alpha-SMA, and fibroblast surface antigen (FSA). Both 2D and 3D cultures demonstrated abundant extracellular matrix formation, including assembly of an elastic fibrillar network, particularly upon addition of Ficoll, which was more pronounced in PEX than in POAG cultures. Expression of PEX-relevant matrix genes, such as fibrillin-1, tropoelastin, MFAP2, LTBP1/2 and LOXL1 were confirmed by immunocytochemistry and qPCR. The presence of typical fibrillar PEX aggregates could be demonstrated by electron microscopy.
Conclusions :
The established in vitro model provides a useful and easily accessable tool for studying disease mechanisms, assessing the impact of genetic and external factors, and exploring effects of targeted therapies for PEX-associated fibrosis.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.