A TGFB2/TNF-induced in vitro model of proliferative vitreoretinopathy (PVR) using ARPE-19 cells confirms nicotinamide as an inhibitor of EMT and VEGFA secretion.

Abstract

Proliferative vitreoretinopathy (PVR) is a vision-threatening fibrotic retinal disorder characterized by the epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells. In this study, we established a pathophysiologically relevant in vitro model by co-stimulating ARPE-19 cells with transforming growth factor beta 2 (TGFB2) and tumor necrosis factor-alpha (TNF), referred to as 'TNT', and evaluated the anti-fibrotic and anti-angiogenic effects of Nicotinamide (NAM), a vitamin B3 derivative previously reported to counteract fibrosis in various disease models. Confluent ARPE-19 cells were treated with TGFB2, TNF, or TNT for up to six days. EMT progression was assessed via immunocytochemistry, Western blotting, and collagen gel contraction assays. Live-cell imaging (LCI) combined with Hoechst 33342 nuclear staining and automated tracking using Fiji/TrackMate enabled real-time analysis of cell migration and multicellular aggregation. VEGFA secretion was quantified by ELISA. TNT stimulation induced synergistic EMT-like features, including cell elongation, directional migration, extracellular matrix (ECM) remodeling, gel contraction, and formation of multicellular aggregates. TrackMate-based analysis revealed coordinated nuclear migration under TNT conditions. VEGFA secretion was significantly elevated at early time points. NAM co-treatment reduced ECM protein expression (FN1, COL1A1), attenuated migration and contraction, and significantly lowered VEGFA release. This TNT-based ARPE-19 model represents a robust, live-cell-compatible in vitro system that mimics both fibrotic and pro-angiogenic aspects of PVR. It allows real-time assessment of EMT progression and is suitable for screening anti-fibrotic compounds. Our findings suggest that Nicotinamide mitigates both fibrotic and angiogenic responses in this model and may hold therapeutic potential for fibrotic retinal diseases.