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Mai T Thao, Mikhail Pavelko, Patrick Wendling, Steven Szlembarski, James P Dillon, Elizabeth R Gaillard; Comparing the Effects of Modified Fibronectin on ARPE-19 Cells: Model Systems of Inflammation and Ageing. Invest. Ophthalmol. Vis. Sci. 2014;55(13):636.
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Aging and inflammation are strongly associated with Age-Related Macular Degeneration (AMD). We have previously identified that these events release molecules that react with intrinsic proteins such as fibronectin. Fibronectin binds to the integrin α5β1 region of the retinal pigment epithelium (RPE) via its amino acid sequence RGD. It is unclear how the modifications to fibronectin by non-enzymatic glycation, A2E mediated blue light damage or non-enzymatic nitration will affect RPE cells. Therefore, in this study the effects of these different modified fibronectins on ARPE-19 cells are compared.
Non-enzymatic glycation and A2E mediated blue light damage to fibronectin were performed to model ageing. Non-enzymatic nitration of fibronectin was also performed to model inflammation. These modified fibronectins were used to coat 24-well plates. For cell attachment assay, 50 x 103 ARPE-19 cells were seeded and allowed to attach for 30 minutes. Unattached cells were removed and attached cells were allowed to grow for 1 hour. Finally, MTT assays were performed to determine cell viability. To determine whether the ARPE-19 cells enter apoptosis or necrosis, 10 x 103 cells were seeded onto modified fibronectin-coated plates. Alexa Fluor 488 was used to determine apoptosis or necrosis with a confocal microscope. Flow cytometry was also used to sort and count cells.
Unmodified fibronectin-coated wells had more ARPE-19 cell attachment compared to the modified fibronectin-coated wells. In addition, nitrated fibronectin had the least cell attachment compared to the glycated or A2E mediated blue light damaged fibronectin. More ARPE-19 cells were observed to be apoptotic than necrotic when they were seeded on the different modified fibronectin-coated wells.
All three methods of modications to fibronectin resulted in less ARPE-19 cell attachment and more apoptosis. Nitration showed the greatest effect. This study demonstrated that modifications to the extracellular matrix proteins can damage RPE viability. Furthermore, it can advance the understanding of AMD pathogenesis.
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