Non-Enzymatic Nitration of Matrigel Deleteriously Effects Retinal Pigment Epithelial Cell Function and Viability

Abstract

Abstract: : Purpose: Post-translational modification of basement membrane proteins can have a profound effect on cell function. Several such studies have been conducted using the non-enzymatic glycation model system. We have previously described the non-enzymatic nitration model system for connective tissue protein damage and have indicated its potential relevance to smoking-related disease. Since smoking has been linked to the development of age-related macular degeneration, we wanted to determine what effects, if any, non-enzymatic nitration of basement membrane-like proteins have on RPE cell function and viability. We have also compared these effects to RPE cells on glycolaldehyde modified basement membrane. Methods: Growth Factor Reduced Matrigel (BD Biosciences) was reacted with NaNO₂ or glycolaldehyde at neutral pH for 7 wks (NaNO₂) or 10 hours (glycolaldehyde) as previously described. Calf RPE cells (passage 4) were then plated at the same cell density on the modified matrices and examined in several ways. Attachment rates, proliferation rates, apoptosis and necrosis were determined and compared to control cultures. Cell morphology was also monitored. Results: RPE cells grown on nitrated and glycated matrigel showed marked differences from controls. Control cells showed typical epithelial morphology while cells on the modified matrices exhibit more fibroblast-like morphology. Cells that were plated onto the nitrite modified matrix appeared enlarged and had significant vacuolization. Attachment rates were reduced for RPE cells on nitrated (two-fold) and glycated (> four-fold). When the cells are plated at low density (1.1 x 10⁴ cells/mL), the proliferation rates are inhibited on both matrices (21% for nitrite modification and 22% for glycolaldehyde modification) but the effect is much more modest when the cells are plated at higher density (5.6 x 10⁴ cells/mL). Conclusions: Non-enzymatic nitration of matrigel has significant damaging effects on RPE cell function and viability that is similar to the damaging effects of glycolaldehyde modification to the basement membrane. These studies may have relevance to the RPE dysfunction/atrophy observed during the progression of AMD.

Keywords: aging • extracellular matrix • retinal pigment epithelium