Cornea - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaROCK inhibitor, phosphorylation of Akt was analyzed for 1 h up to24 h in serum-containing medium: in the absence of ROCK inhibitor,phosphorylation of Akt was gradually increased to 12 h. Unlike thecontrol pattern, phosphorylation of Akt reached maximum at 1 hfollowing treatment of cells with ROCK inhibitor, after which thelevel of phosphorylated Akt was gradually decreased. The expressionof Cdc25A, which activates Cdk2 that phosphorylates p27, wasobserved during the early time periods in the presence of ROCKinhibitor, while the control cells showed a late expression pattern ofCdc25A. Similarly, the amount of p27 was greatly reduced from 1 hfollowing treatment of cells with ROCK inhibitor, whereas thecontrol cells maintained high levels of p27 up to 12 h.Conclusions: The findings of the study demonstrate that ROCKinhibitor activates PI 3-kinase signaling which subsequentlypromotes degradation of p27 via Cdc25A pathway, thus leading tocell proliferation of CECs. These data suggest that the ROCK mightbe a useful pharmaceutical agent for corneal endothelial disease.Commercial Relationships: Ryohei Numata, None; NaokiOkumura, None; EunDuck P. Kay, None; Makiko Nakahara,None; Shinichiro Nakano, None; Morio Ueno, None; ShigeruKinoshita, Senju Pharmaceutical Co (P), Santen Pharmaceutical Co(P), Otsuka Pharmaceutical Co (C), Alcon (R), AMO (R), HOYA(R); Noriko Koizumi, NoneSupport: The Adaptable and Seamless Technology TransferProgram through Target-driven R&D (AS2314212G),The FundingProgram for Next Generation World-Leading Researchers from theCabinet Office in Japan ( LS117)Program Number: 1691 Poster Board Number: D0326Presentation Time: 8:30 AM - 10:15 AMProliferation Propensity of Cultured Human Corneal EndothelialCells and Their Plasticity Dictated by CultureMicroenvironmentsMunetoyo Toda 1 , Kana Nakata 1 , kazuko asada 1 , Michio Hagiya 1 ,Morio Ueno 1 , Naoki Okumura 2 , Noriko Koizumi 2 , Junji Hamuro 1 ,Shigeru Kinoshita 1 . 1 Department of Ophthalmology, KyotoPrefectural University of Medicine, Kyoto, Japan; 2 Department ofBiomedical Engineering, Doshisha University, Kyoto, Japan.Purpose: It is well known that human corneal endothelium cells(HCECs) have poor proliferative ability under in vitro cultureconditions. The difficulty of cultivating HCECs hampers a detailedanalysis of their proliferation propensity. To detail molecularmechanisms underlying this impaired HCEC proliferation, weattempted to clarify the presence and proliferation propensity offunctionally heterogeneous subpopulations in cultured HCECs.Methods: The proliferative properties of cultured HCECs wereevaluated by BrdU assay and carboxyfluorescein succinimidyl ester(CFSE) dye dilution assay. To investigate if cultured HCECs containsubpopulations with distinct metabolic requirements, HCECs werestained with MitoTracker® Red (Life Technologies Corp., Carlsbad,CA) to evaluate their mitochondria content. Flow cytometry (FCM)using several surface markers was performed to characterize thesubpopulations.Results: Cell percentages in the G1, S, and G2/M phase of the cellcycle were determined by FCM using BrdU and 7-AAD. Thepercentage of cells in the S- and G2/M-phase was about 40%, andabout 60% of the HCECs were arrested in the G1-phase. CFSE assaydetected 2 subpopulations with different proliferative properties. Onedivided 7 times in 8 days of cultivation, while the other stopped celldivision at 3 times. We theorize that the latter entered premature cellsenescence at the early stage of cultivation, resulting in cell-cyclearrest. These results suggest that each subpopulation has uniquemetabolic turnover rates and energy requirements, as it is theorizedthat poor proliferation is tied to energy from mitochondria, not fromglucose metabolism. Moreover, FMC using MitoTracker® Reddetected different mitochondrial content in the cultured HCECs. Eachsubpopulation was stained with several cell surface markers selectedby global analysis, and we tried to characterize candidate markers forthe high-proliferative population.Conclusions: These findings show that cultured HCECs havedifferent subpopulations and provide the possibility of establishing aneffective method for culturing HCECs containing an enrichedsubpopulation with high proliferation ability.Commercial Relationships: Munetoyo Toda, None; Kana Nakata,None; kazuko asada, None; Michio Hagiya, JCR PharmaceuticalsCo., Ltd (E); Morio Ueno, None; Naoki Okumura, None; NorikoKoizumi, None; Junji Hamuro, None; Shigeru Kinoshita, SenjuPharmaceutical Co (P), Santen Pharmaceutical Co (P), OtsukaPharmaceutical Co (C), Alcon (R), AMO (R), HOYA (R)Program Number: 1692 Poster Board Number: D0327Presentation Time: 8:30 AM - 10:15 AMCell-injection Therapy Using Rho Kinase Inhibitor in a CornealEndothelial Dysfunction Rabbit ModelJunji Kitano 1 , Naoki Okumura 1, 2 , EunDuck P. Kay 1 , Morio Ueno 2 ,Junji Hamuro 2 , Shigeru Kinoshita 2 , Noriko Koizumi 1, 2 . 1 BiomedicalEngineering, Doshisha University, Kyotanabe, Japan;2 Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto,Japan.Purpose: To investigate feasibility of corneal endothelialreconstruction by a cell-injection therapy using cultivated rabbitcorneal endothelial cells (RCECs) in a corneal endothelialdysfunction rabbit model and to determine the optimum cell numbersfor the cell-injection therapy.Methods: Rabbit corneal endothelium was denuded by intensivemechanical scraping. Cultivated RCECs in the presence of 100μM ofRho kinase (ROCK) inhibitor, Y-27632, were injected into theanterior chamber of the host animals at three concentrations (2x10 5cells, 5.0x10 5 cells, or 1.0x10 6 cells). The eyes of each animal werekept in the face-down position for 3 hours. Slit-lamp examinations,corneal thickness- and intraocular pressure- measurements, andimmunohistochemical analysis were performed for up to 14 days.Results: All eyes received cell-injection therapy showed improvedcorneal clarity; corneal clarity and corneal thickness recovered thefastest rate in the host animals that received cultivated RCECs at1.0x10 6 cell numbers. In all animal groups, corneal endotheliumdemonstrated the characteristic contact-inhibited monolayer withpolygonal cells that express the functional endothelial phenotypicproteins, ZO-1 and Na+/K+-ATPase. When the endothelial celldensity of the host animals was measured, the animal that received acell injection at 1.0x10 6 cells demonstrated significant high celldensity with 3296.6±365.1 cells/mm 2 , while the other two animalgroups showed 1432.0±200.8 cells/mm 2 (injected cell numbers:2.0x10 5 ) and 2252.2±204.6 cells/mm 2 (injected cell numbers: 5.0x10 5cells). None of the eyes of the experimental animals showed elevatedintraocular pressure or immunological rejection.Conclusions: The findings provide evidence that the cell-injectiontherapy using appropriate cell numbers with ROCK inhibitor enablescorneal endothelial regeneration by tissue engineering technique andmay be a useful clinical alternative for corneal transplantation.Commercial Relationships: Junji Kitano, None; Naoki Okumura,None; EunDuck P. Kay, None; Morio Ueno, None; Junji Hamuro,None; Shigeru Kinoshita, Senju Pharmaceutical Co (P), SantenPharmaceutical Co (P), Otsuka Pharmaceutical Co (C), Alcon (R),AMO (R), HOYA (R); Noriko Koizumi, None©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.