PurposeTo investigate the expression of humanin (HN) in human retinal pigment epithelial (hRPE) cells and its effect on oxidative stress–induced cell death, mitochondrial bioenergetics, and senescence.MethodsHumanin localization in RPE cells and polarized RPE monolayers was assessed by confocal microscopy. Human RPE cells were treated with 150 μM tert-Butyl hydroperoxide (tBH) in the absence/presence of HN (0.5–10 μg/mL) for 24 hours. Mitochondrial respiration was measured by XF96 analyzer. Retinal pigment epithelial cell death and caspase-3 activation, mitochondrial biogenesis and senescence were analyzed by TUNEL, immunoblot analysis, mitochondrial DNA copy number, SA-β-Gal staining, and p16INK4a expression and HN levels by ELISA. Oxidative stress–induced changes in transepithelial resistance were studied in RPE monolayers with and without HN cotreatment.ResultsA prominent localization of HN was found in the cytoplasmic and mitochondrial compartments of hRPE. Humanin cotreatment inhibited tBH-induced reactive oxygen species formation and significantly restored mitochondrial bioenergetics in hRPE cells. Exogenous HN was taken up by RPE and colocalized with mitochondria. The oxidative stress–induced decrease in mitochondrial bioenergetics was prevented by HN cotreatment. Humanin treatment increased mitochondrial DNA copy number and upregulated mitochondrial transcription factor A, a key biogenesis regulator protein. Humanin protected RPE cells from oxidative stress–induced cell death by STAT3 phosphorylation and inhibiting caspase-3 activation. Humanin treatment inhibited oxidant-induced senescence. Polarized RPE demonstrated elevated cellular HN and increased resistance to cell death.ConclusionsHumanin protected RPE cells against oxidative stress–induced cell death and restored mitochondrial function. Our data suggest a potential role for HN therapy in the prevention of retinal degeneration, including AMD.

Citation: He S, Barron E, Ishikawa K, et al. Inhibition of DNA methylation and methyl-CpG-binding protein 2 suppresses RPE transdifferentiation: relevance to proliferative vitreoretinopathy. Invest Ophthalmol Vis Sci. 2015;56:5579-5589. DOI:10.1167/ iovs.14-16258 PURPOSE. The purpose of this study was to evaluate expression of methyl-CpG-binding protein 2 (MeCP2) in epiretinal membranes from patients with proliferative vitreoretinopathy (PVR) and to investigate effects of inhibition of MeCP2 and DNA methylation on transforming growth factor (TGF)-b-induced retinal pigment epithelial (RPE) cell transdifferentiation. METHODS.Expression of MeCP2 and its colocalization with cytokeratin and a-smooth muscle actin (a-SMA) in surgically excised PVR membranes was studied using immunohistochemistry. The effects of 5-AZA-2 0 -deoxycytidine (5-AZA-dC) on human RPE cell migration and viability were evaluated using a modified Boyden chamber assay and the colorimetric 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay. Expression of RASAL1 mRNA and its promoter region methylation were evaluated by real-time PCR and methylationspecific PCR. Effects of 5-AZA-dC on expression of a-SMA, fibronectin (FN), and TGF-b receptor 2 (TGF-b R2) and Smad2/3 phosphorylation were analyzed by Western blotting. Effect of short interfering RNA (siRNA) knock-down of MeCP2 on expression of a-SMA and FN induced by TGFb was determined.RESULTS. MeCP2 was abundantly expressed in cells within PVR membranes where it was double labeled with cells positive for cytokeratin and a-SMA. 5-AZA-dC inhibited expression of MeCP2 and suppressed RASAL1 gene methylation while increasing expression of the RASAL1 gene. Treatment with 5-AZA-dC significantly suppressed the expression of a-SMA, FN, TGF-b R2 and phosphorylation of Smad2/3 and inhibited RPE cell migration. TGF-b induced expression of a-SMA, and FN was suppressed by knock-down of MeCP2.CONCLUSIONS. MeCP2 and DNA methylation regulate RPE transdifferentiation and may be involved in the pathogenesis of PVR.

Proliferative vitreoretinopathy (PVR) is the most common cause for failure of retinal reattachment surgery. In a search for better pharmacologic treatment of PVR, we investigated the effect of intravenous injections of suramin on an experimental rabbit model of PVR. PVR was induced in rabbits by intravitreal injection of autologous fibroblasts. The experimental group (7 eyes) received intravenous injections of suramin (100 mg/kg body weight) every 3 days for 15 days, beginning 3 days before fÍbroblast injection. The control group (5 eyes) was treated similarly but received intravenous saline solution in place of suramin. A third group (4 eyes) received suramin according to the protocol above but did not receive intravitreal fibroblasts. The animals were examined by indirect ophthalmoscopy every 3 days and were sacrificed 14 days after the injection of fibroblasts. The serum levels of suramin were evaluated by high-performance liquid chromatography. The PVR was classified as stages I-V, based upon clinical findings. PVR developed in both experimental and control animals but was less severe in those treated with suramin. On day 14, the average stage of PVR in the control group was 3.8; in the suramin-treated group, however, the average stage was 2.4, which was significantly less than in the control group (p < 0.02). None of the rabbits in the third group showed pathologic changes. Serum levels of suramin were maintained at an average of 280.2 µg/ml and no apparent toxicity was found in the retina by histologic study. These results suggest that suramin has an inhibitory effect on PVR in a rabbit model and may have the potential as a pharmacologic modulator of PVR in humans.

Mitochondrial dysfunction and oxidative stress are thought to be relevant to the pathogenesis of age-related macular degeneration (AMD). Glutathione (GSH) homeostasis fulfills a number of important roles in mitochondria, such as maintenance of mitochondrial DNA and respiratory competency of cells. Although the transport of mitochondrial GSH (mGSH) is not fully understood, increasing evidence from non-ocular tissues suggests that OGC (2-oxoglutarate carrier, SLC25A11) and DIC (dicarboxylate carrier, SLC25A10) are involved in mGSH transport. However, whether OGC and DIC mediate the transfer of GSH into the mitochondria of retinal pigment epithelial cells (RPE) remains unknown. Thus, we investigated the expression, localization, and function of OGC and DIC in human RPE (hRPE) in relation to oxidative stress and GSH. Both OGC and DIC are expressed in hRPE and are localized in mitochondria. We also found a dose and time-dependent decrease of OGC and DIC expression under oxidative stress and increased expression in polarized RPE. Our data show that the downregulation of OGC and DIC resulted in increased apoptosis and mGSH depletion which can be overcome by co-treatment with GSH-MEE. These findings suggest that overexpression of OGC and DIC may be an effective strategy to decrease susceptibility to mitochondrial toxicants by elevation of mGSH.