Cornea - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaART with undetectable viral load, CDKN2A expression and 8-OHDG levels were higher in those with accelerated aging, asreflected by lower ECD.Conclusions: The corneal endothelium shows features consistentwith HIV-related accelerated senescence, especially among thosewith poor immune recovery.Commercial Relationships: Sophia Pathai, None; Stephen D.Lawn, None; Paul G. Shiels, Pathfinder Cell Therapy (F), SanofiAventis (C); Helen A. Weiss, None; Colin Cook, None; RobinWood, None; Clare E. Gilbert, NoneSupport: Wellcome Trust 090354/Z/09/ZProgram Number: 2201Presentation Time: 4:00 PM - 4:15 PMEfficacy and Safety Evaluation of Cell-Injection Therapy usingCultivated Human Corneal Endothelial CellsNoriko Koizumi 1, 2 , Naoki Okumura 1, 2 , Takashi Shiina 3 , ShingoSuzuki 3 , Shinichiro Nakamura 4 , Yuji Sakamoto 1 , Kenta Yamasaki 1 ,Morio Ueno 2 , Junji Hamuro 2 , Shigeru Kinoshita 2 . 1 BiomedicalEngineering, Doshisha University, Kyotanabe City, Japan;2 Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto,Japan; 3 Molecular Life Sciences, Tokai University School ofMedicine, Isehara, Japan; 4 Research Center for Animal Life Science,Shiga University of Medical Science, Otsu, Japan.Purpose: To investigate the efficacy and safety of cornealendothelial reconstruction by a cell-injection therapy using cultivatedhuman corneal endothelial cells (HCECs) in a corneal endothelialdysfunction monkey model.Methods: The corneal endothelium of 8 eyes of 8 monkeys wasintensively scraped off up to the peripheral area to create a cornealendothelial dysfunction model. A 5.0 x 10 5 amount of cultivatedHCECs, labeled by fluorescein marker DiI, was then injected into theanterior chamber of those 8 eyes. Slit-lamp examinations andintraocular pressure (IOP) measurements of the cell-injected eyeswere then performed on postoperative day 1 through day 14.Fourteen days after cell-injection, 31 primary organs were harvestedfrom each animal after euthanasia and were set aside for histologicalexamination. To examine the ectopic deposition of donor HCECssystemically, tissue sections obtained from those 31 organs wereexamined by fluorescein microscopy to search for DiI-positive donorcells. Genomic DNA was then extracted from tissue samples of 31organs and examined by polymerase chain reaction (PCR) usinghuman-specific primers to elucidate if it was contaminated with anydonor-HCEC-derived DNA.Results: Seven days after cell-injection, 6 of the 8 eyes (75%)recovered corneal clarity. No undesirable accumulation of HCECs inocular tissue, elevation of IOP, or systemic side-effects was observed.Histological examination of the 31 organ-tissue samples revealed notumor genesis or inflammatory response. Moreover, PCRexamination of those samples revealed no genomic DNA derivedfrom the donor HCECs.Conclusions: The findings of this present study indicate that cellinjectiontherapy using cultivated HCECs is a safe and effectiveprocedure, and one that might be clinically applicable for thetreatment of corneal endothelial dysfunction.Commercial Relationships: Noriko Koizumi, None; NaokiOkumura, None; Takashi Shiina, None; Shingo Suzuki, None;Shinichiro Nakamura, None; Yuji Sakamoto, None; KentaYamasaki, 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)Support: The Funding Program for Next Generation World-LeadingResearchers from the Cabinet Office in Japan ( LS117)Program Number: 2202Presentation Time: 4:15 PM - 4:30 PMActivation of the Rho/ROCK Signaling Pathway in the Apoptosisof Corneal Endothelial CellsNaoki Okumura 1, 2 , Ai Odajima 1, 2 , EunDuck P. Kay 1 , Wen Chen 1 ,Morio Ueno 2 , Junji Hamuro 2 , Shigeru Kinoshita 2 , Noriko Koizumi 1 .1 Biomedical Engineering, Doshisha University, Kyotanabe, Japan;2 Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto,Japan.Purpose: In the pathological condition of corneal endotheliumassociated with Fuchs’ dystrophy or post keratoplasty, apoptosis isknown to be involved. We previously reported that Rho kinase(ROCK)-inhibitor Y-27632 suppresses the apoptosis of culturedcorneal endothelial cells (CECs). The purpose of this present studywas to evaluate the involvement of the Rho/ROCK signaling pathwayin the apoptosis of CECs and the effect of ROCK inhibitor onmodulating apoptosis.Methods: Monkey corneal endothelial cells (MCECs) were culturedand then exposed to ultra-violet (UV) radiation (100J/m 2 ) to induceapoptosis. To elucidate the involvement of the Rho/ROCK signalingpathway in apoptosis, RhoA-GTPase, myosin light chain (MLC)phosphorylation, and the cleavage of caspase 3 and ROCK 1 wasevaluated by western blotting. Anti-blebbing effect of Y-27632 wasevaluated after exposure to UV radiation by time-lapse phase contrastmicroscopy, and actin and MLC phosphorylation were evaluated byimmunostaining. Annexin V staining was employed to confirm theanti-apoptotic effect of Y-27632.Results: UV radiation caused cell death in MCECs in a dosedependentmanner; a significant cell death was observed at100J/m 2 and higher dosages. Active GTPase pull-down assay showedthat UV radiation activated RhoA of the MCECs. MLCphosphorylation and the cleavage of caspase 3 and ROCK 1 wereincreased by UV radiation. Compared to the control, Y-27632-treatedMCECs exposed to UV radiation showed significantly decreasedmembrane blebbing (p