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Hiroki Hatanaka, Atsushi Mukai, Kazuhito Yoneda, Jun Yamada, Chie Sotozono, Shigeru Kinoshita, Junji Hamuro; Effect of epigenetic regulation on fibrotic change in retinal pigment epithelium cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6530. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Proliferative eye diseases such as proliferative vitreoretinopathy and proliferative diabetic retinopathy are a major cause of blindness. Age-related macular degeneration (AMD) is recently on the increase, and it causes central scotoma and severe reduced visual acuity through the progression of choroidal neovascularization and the formation of fibrin at the macula area. Fibroblastic change of retinal pigment epithelium (RPE) cells or the fibroblastic membrane is one of the major causes of proliferative eye diseases. The purpose of this present study was to examine the effect of histone deacetylase (HDAC) and histone acetyl transferase (HAT) on fibrotic change in RPE cells.
Following the method described in previous reports, ARPE-19 cells (5×105 cells/mm2) were cultured in10% fetal bovine serum (FBS) and then starved in serum-free medium for 24 hours. The culture medium was then replaced with fresh transforming growth factor beta 2 (TGF-β2, 30ng/ml) or/and tumor necrosis factor alpha (TNF-α, 10ng/ml) medium, and fibrotic change of the ARPE-19 cells was induced. Culture medium without TGF-β2 and TNF-α was used as a control. Cell morphology was examined by phase contrast microscopy after 48 hours of incubation. Simultaneously, the expression of α-smooth muscle actin (α-SMA), matrix metalloproteinase-9 (MMP-9), and CD44 were examined by immunostaining, western blotting, and polymerase chain reaction (PCR). The activity of HDAC and HAT were then observed between the following four groups: 1) control, 2) TGF-β2, 3) TNF-α, and 4) TGF-β2+TNF-α.
Fibroblastic change of ARPE-19 cells was observed in all groups except for the control, and the potentiating effect of the change was noted in the medium with TGF-β2+TNF-α. The expression of α-SMA mRNA was enhanced by TGF-β2 (p<0.05), yet reduced by TNF-α or TGF-β2+TNF-α (p<0.01). The MMP-9 and CD44 mRNA expression was increased by TNF-α or TGF-β2+TNF-α (p<0.01). Similar trends were observed in immunostaining and western blotting. The activity of HAT was reduced by TNF-α or TGF-β2+TNF-α (p<0.05), and that of HDAC1 was significantly increased by TGF-β2+TNF-α (p<0.01).
Our findings indicate that alterations in the activity of HDAC and HAT are likely related to the fibroblastic change of RPE cells. We speculate that HDAC and HAT will become therapeutic targets for proliferative eye diseases and AMD.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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