Cornea - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaform stable crosslinked products and is an effective crosslinkingagent for Descemet membrane fixation. Therefore, these resultsindicated that the genipin-fixed Descemet membrane may be appliedas a excellent culture carrier. The non-toxic nature crosslinkergenipin may be useful for the application on the reconstruction of theocular surface.Commercial Relationships: Lung-Kun Yeh, None; Shih-ChunHuang, NoneSupport: Chang Medical Research Project G CMRPG3A1291 ;National Science Council Grants (Taiwan) 1012314B182A056MY3Program Number: 542 Poster Board Number: B0179Presentation Time: 10:30 AM - 12:15 PMThe Role of Nrf2-Mediated Defense System in Corneal EpithelialWound HealingRyuhei Hayashi 1 , Noriko Himori 2 , Keiko Taguchi 3 , Yuki Ishikawa 1 ,Kohji Uesugi 1 , Motokazu Tsujikawa 1 , Toru Nakazawa 2 , MasayukiYamamoto 3 , Kohji Nishida 1 . 1 Ophthalmology, Osaka UniversityMedical School, Suita, Japan; 2 Ophthalmology, Tohoku universityschool of medicine, Sendai, Japan; 3 Department of MedicalBiochemistry, Tohoku university school of medicine, Sendai, Japan.Purpose: The Nrf2-mediated defense system plays a central role inprotecting cells by activating genes against these types of stress. Inthe present study, we investigated the role of the Nrf2-mediateddefense system in corneal epithelial wound healing by using Nrf2-knockout (KO) mice.Methods: The corneal epithelium of wild type (WT) and Nrf2 KOmice were removed by treatment of n-heptanol for 1 minute underanesthesia. The epithelial defect was stained with 1% fluoresceinsolution and photographed at 0, 6, 12, 18, 24, 30, 36, 48, 60, and 72 hafter epithelial debridement. Injured corneas healed at various timepoints were subjected to immunohistochemistry with Ki-67and Nrf2antibody. Telomerase-immortalized corneal epithelial cell line(C/TERT) was used for cell migration assay and siRNA experiment.Results: Nrf2 was expressed in the corneal epithelium of WT mice,but not in KO mice. Observation of wounds after 24 h of healingrevealed that healing of the corneal epithelium was significantlydelayed in the Nrf2 KO mice, while Nrf2 was activated in the cornealepithelium of WT mice. Ki-67-staining revealed that the number ofKi-67-positive proliferation cells was significantly lower in the Nrf2KO mice than in the WT mice at 24-36 h after injury; however, thesenumbers were approximately equivalent by 48 h. To clarify the roleof Nrf2 during wound healing, we performed in vitro experiments ofsiRNA for Nrf2. The result showed that Nrf2 knock-downsignificantly delayed corneal epithelial cell migration.Conclusions: This study provides evidence that the Nrf2-mediateddefense system plays a crucial role in corneal epithelial woundhealing, by regulating the cell-migration activities of cornealepithelial cells.Commercial Relationships: Ryuhei Hayashi, None; NorikoHimori, None; Keiko Taguchi, None; Yuki Ishikawa, None; KohjiUesugi, None; Motokazu Tsujikawa, Shionogi & Co. (C), DaiichiSankyo Co. (F), Daiichi Sankyo Co. (R), Santen Co. (R), AMO Co.(R); Toru Nakazawa, Kowa Company Ltd. (F), Kowa CompanyLtd. (C); Masayuki Yamamoto, None; Kohji Nishida, Alcon (C),Alcon (F), HOYA (F), Senju (F), Pfizer (F), Santen (F), OsakaUniversity (P)Support: Grants-in-Aid for Scientific Research from the Ministry ofEducation, Culture, Sports, Science and Technology in JapanProgram Number: 543 Poster Board Number: B0180Presentation Time: 10:30 AM - 12:15 PMIncreased fragility and acceleration of migration of cornealepithelial cells in an epiplakin deficiencyMasahide Kokado 1 , Yuka Okada 1 , Kazushi Ishikawa 2 , HiromitsuShimada 2 , Sakuhei Fujiwara 2 , Masayasu Miyajima 3 , Shizuya Saika 1 .1 Department of Ophthalmology, Wakayama Medical University,Wakayama, Japan; 2 Department of Dermatology, Oita University,Oita, Japan; 3 Laboratory animal center, Wakayama MedicalUniversity, Wakayama, Japan.Purpose: Epiplakin is one of intermediate filament-relatedcomponents. To examine fragility and mechanism of migration ofcorneal epithelial cells in an epiplakin deficiency. We previouslyreported that the loss of epiplakin facilitates healing of a defect incorneal epithelium in mice (ARVO 2009), and epiplakin knockdownby Short-interfering RNA (siRNA) was accelerated the migration ofHCEC and suppressed TGFb1 activation of p38 and JNK signals(ARVO2012).Methods: (1) We used Epiplakin KO (KO) (n=4) and wild type(WT) (n=4). The corneal surface was gently brushed with a surgicalmicro-sponge. Then ultrathin sections were cut and observed undertransmission electron microscopy. (2) We ran real-time RT-PCR forcell-cell connection-related components in RNA samples obtainedfrom KO (n=12)and WT (n=12). (3)Effect of siRNA knockdown ofEPPK on E-cadherin expression was also studied in AV40-immortalized corneal epithelial cells by using western blotting.Results: (1)Transmission electron microscopy showed that eachlayer of the epithelial cells was well maintained following brushing ina WT epithelium although partial separation between cells of thesuperficial layer. In a KO epithelium one or two layer(s) of basal orsupra-basal epithelial cells were found to be in the original positionfollowing the treatment and upper layer cells were found to beremoved. (2)Real-time RT-PCR also showed that mRNA expressionof E-cadherin was suppressed by the loss of epiplakin, while that ofdesmoglein-1 or desmoplakin-1 was not affected by epiplakin geneknockout in a mouse cornea. (3)EPPK knockdown suppressed E-cadherin expression in the cells.Conclusions: The loss of epiplakin affects the corneal epitheliumintegrity. The mechanism of acceleration of cell migration in the KOcorneal epithelium is to be further investigated, although suppressionof expression of E-cadherin in the absence of EPPK might beincluded. The possibility of the potential corneal epitheliumdisturbance in the patient who has abnormalities to EPPK wassuggested.Commercial Relationships: Masahide Kokado, None; YukaOkada, None; Kazushi Ishikawa, None; Hiromitsu Shimada,None; Sakuhei Fujiwara, None; Masayasu Miyajima, None;Shizuya Saika, NoneProgram Number: 544 Poster Board Number: B0181Presentation Time: 10:30 AM - 12:15 PMTransfer of mucosal epithelial cells and extracellular matrix by agelatin matrix technique in vitroAllen Ho 1 , Li-Fang Wang 2 . 1 Jianguo School, Taipei, Taiwan;2 National Taiwan University, Taipei, Taiwan.Purpose: Poor connection and adhesion of epithelial cells tounderlying connective tissues is the basic defect of blister disease ofconjunctiva, skin, and mouth mucosal membrane such as ocularcicatricial pemphigoid. Transplantation of extracellular matrix(ECM) along with the autologous buccal mucosal epithelial cells maymodulate the survival and subsequent proliferation of transplantedepithelial cells. Thus, we have developed a technique to transfernative ECM with human mucosal epithelial cells in vitro.Methods: Confluent monolayer human mucosal epithelial cells in theculture plate was pretreated with 0.25% edetic acid for 15 minutes©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.