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Whitney Greene; The development of an in vitro model of proliferative vitreoretinopathy using retinal pigment epithelium derived from induced pluripotent stem cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1274.
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© ARVO (1962-2015); The Authors (2016-present)
To study the mechanisms that underlie the three phases of proliferative vitreoretinopathy (PVR): migration, proliferation, and contraction. Retinal pigment epithelium derived from induced pluripotent stem cells (iPS-RPE) provides a physiologically relevant in vitro model to study the cellular events that drive the pathology of PVR.
iPS-RPE was grown to confluency on transwells. The cell monolayer was scratched to create a wound. Conditioned media from the cells was collected for ELISA analysis of cytokines and growth factors. iPS-RPE was also treated with 5% vitreous fluid, 5% whole blood, or vitreous fluid plus blood. At 21 days after wounding, the iPS-RPE were fixed and labeled for smooth muscle actin and b-catenin. Western blot analysis was performed to detect expression of proteins that regulate epithelial-mesenchymal transition (EMT). Cell proliferation was measured after treatment with vitreous and blood.
Following wounding of the monolayer, iPS-RPE demonstrated polarized secretion of PDGF and VEGF. Cells treated with vitreous fluid, whole blood, or vitreous fluid plus blood demonstrated higher rates of migration and proliferation into the wound area compared to untreated cells. In addition, vitreous fluid induced contraction of the wound area leading to detachment of the cell monolayer. Immunofluorescence analysis revealed translocation of b-catenin to the nucleus. Expression of smooth muscle actin increased after exposure to vitreous and blood. Increased cell proliferation after exposure to vitreous and blood was confirmed. Western blot analysis revealed that treatment with vitreous fluid and blood induced the phosphorylation of SMAD2/3 and increased the expression of smooth muscle actin, SNAIL, SLUG, and SRF proteins.
Exposure to vitreous fluid and blood induced increased cell migration, proliferation, and contraction. The genes that regulate EMT were upregulated following treatment with vitreous and blood. The increased expression of smooth muscle actin indicates the cells have transdifferentiated into myofibroblasts, the cells that provide the contractile force and cause the secondary retinal detachment that occurs in PVR. These results indicate that iPS-RPE provide a physiologically relevant model that can be used to screen pharmacological compounds for the ability to inhibit the pathogenesis of PVR.
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