Abstract
Purpose :
Age-related macular degeneration (AMD) is a complex and progressive disease of the macula that mainly affects the elderly population. Aging causes thickening and ultrastructural changes in the extracellular matrix (ECM) composition of Bruch’s membrane (BrM) leading to changes in retinal pigment epithelium (RPE) cells. Recent studies strongly suggest that the major risk for AMD is linked to genetic variants in the ARMS2 gene, particularly the ARMS2-69S variant. Previously, we found that ARMS2 functionally interacts with ECM proteins, which are also involved in the development of other maculopathies. The present study investigates, how ARMS2-A69S influences the production, structural organization, and composition of ECM components in RPE cells.
Methods :
The impact of ARMS2 variants was analysed in hTERT-RPE1 cells by overexpression of ARMS2-69A (low risk), ARMS2-69S (high risk), and an empty plasmid control. The changes in ECM deposition were monitored via immunostaining of ECM generated by RPE cells overexpressing ARMS2 constructs followed by decellularization of transwell membranes after four weeks of culture (n=3 for fibronectin, laminin, elastin, and Fibulin-3 staining). Moreover, the levels of ECM components (fibronectin, laminin, elastin, fibulins) and ECM regulatory factors (MMPs and TIMPs) were assessed via Western Blot and qPCR (mean±SEM, n=6).
Results :
RPE cells bearing the ARMS2-69S variant displayed alterations in ECM composition, accumulation, and disorganization of ECM proteins, compared to the ARMS2 low risk variant. In detail, the network of fibronectin fibers appeared homogenous in low-risk ARMS2 RPE cells and fragmented in the high-risk RPE cells. The laminin, elastin, and Fibulin-3 staining displayed extracellular aggregates in the ECM deposited by ARMS2-69S RPE cells. Overexpression of ARMS2-69S led to a significant downregulation of Fibulin-3 gene (0.25 ± 0.2, p<0.05). At the protein level, we observed a decrease in secreted MMP2 (5.2 ± 1.4, p<0.05) and TIMP2 (7.3 ± 1.9, p<0.05) in the supernatant of RPE cells overexpressing ARMS2-69S.
Conclusions :
Our data show that RPE cells produce altered ECM when expressing the ARMS2-A69S variant. Our results confirm a role of ARMS2 in ECM regulation and suggest a link between the ARMS2-A69S polymorphism in RPE cells and ECM changes found in AMD patients.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.