Cornea - ARVO

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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneapatients with FECD.Methods: Human corneal endothelial cells (HCECs) were culturedfrom endothelial-Descemet membrane lamellae obtained from 3FECD patients during Descemet's membrane endothelial keratoplasty(DMEK) after obtaining informed written consent from each patient.HCECs cultured from 3 individual normal donor corneas were usedas a control. The FECD-derived HCECs and the control HCECs werethen immortalized by SV40 and hTERT to produce iFECD andiHCEC cell lines, respectively. The gene expression levels of ECMcomponents were then analyzed by TaqMan® real-time PCR. Toelucidate ECM production, iFECD and iHCEC cells were cultured ina Transwell® culture system, and ECM deposition was then analyzedby hematoxylin-eosin (HE) staining and immunohistochemistry after2 weeks.Results: Real-time PCR revealed a significantly increased productionof type I collagen, type IV collagen, and fibronectin (p
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaAna G. Alzaga Fernandez, None; Kimberly C. Sippel, None;Jessica Ciralsky, None; Mark Rosenblatt, None; Priyanka Sood,NoneSupport: Research to Prevent BlindnessProgram Number: 1683 Poster Board Number: D0318Presentation Time: 8:30 AM - 10:15 AMClinical Manifestation and Effect of Ganciclovir Therapy forCytomegalovirus Corneal EndotheliitisTsutomu Inatomi 1 , Noriko Koizumi 2 , Yuichi Ohashi 3 , YoshitsuguInoue 4 , Manabu Mochizuki 5 , Kohji Nishida 6 . 1 Ophthalmology, KyotoPrefectural Univ of Med, Kamigyo-Ku, Japan; 2 BiomedicalEngineering Faculty of Life and Medical Sciences, DoshishaUniversity, Kyoto, Japan; 3 Ophthalmology, Ehime University Schoolof Medicine, Ehime, Japan; 4 Ophthalmology, Tottori University,Faculty of Medicine, Tottori, Japan; 5 Ophthalmology, Tokyo Medicaland Dental University Graduate School, Tokyo, Japan;6 Ophthalmology, Osaka University Graduate School of Medicine,Osaka, Japan.Purpose: To report the findings of an epidemiological surveyconducted in Japan regarding the clinical manifestations and theeffect of ganciclovir antiviral therapy for the treatment of patientswith cytomegalovirus (CMV) corneal endotheliitis.Methods: An epidemiological survey was conducted in Japan andthe clinical manifestations of 109 eyes of 106 cases diagnosed asCMV corneal endotheliitis were analyzed retrospectively.Prospective, multicenter clinical treatment was performed for 7 eyesof 7 cases. The mean patient age was 67.4±10.4 years, and the meanfollow-up period was 12 months. The combined therapy of systemicganciclovir (5mg/kg, 2 times daily) with topical 0.5% ganciclovirinstillation was applied for 2 weeks following the tapering of topicalinstillation. Clinical evaluation was performed by use of real-timepolymerase chain reaction (PCR) and slit-lamp examination.Results: Of the total 106 cases, 85 cases (80.2%) were male and 21cases (19.8%) were female. Coin-shaped lesions and linear keraticprecipitates (KPs) were observed in 70.6% and 8.3% of the cases,respectively. Clinical manifestations included corneal edema(73.4%), endothelial loss (81.7%), iritis (67.9%), and elevation ofintraocular pressure (66.1%). In the prospective study, the averageCMV viral load in aqueous humor before treatment was 8.55x104copies/ml (range: 8.3x102 - 1.55x105 copies/ml). Coin-shapedlesions or linear KPs were detected in 100% and 29% of the cases,respectively, and 85.7% of the cases showed sustained virologicresponse after the initial combined therapy. All cases showednegative by PCR with additional topical ganciclovir therapy, but 3cases (42.8%) showed the elevation of intraocular pression and 1 case(14.5%) showed the recurrence. Mean endothelial cell density (CED)(cell/mm2 ) at before and 1-month post treatment was 1160±365 and1048±440, respectively. No significant reduction of CED and noadverse effects were observed.Conclusions: Corneal endotheliitis with coin-shaped lesions or linearKPs, similar to the clinical rejection line, are major clinicalmanifestations in CMV corneal endotheliitis. The combined therapyof systemic and topical ganciclovir instillation is safe and effectivefor the treatment of patients with CMV corneal endotheliitis.Commercial Relationships: Tsutomu Inatomi, None; NorikoKoizumi, None; Yuichi Ohashi, None; Yoshitsugu Inoue, None;Manabu Mochizuki, Santen (F), Senju (F), Ohtsuka (F), Daiichi-Sankyo (F), Mitsubishi-Tanabe (F), AMO Japan (F), Alcon Japan(F); Kohji Nishida, Alcon (C), Alcon (F), HOYA (F), Senju (F),Pfizer (F), Santen (F), Osaka University (P)Support: Research on Measures for Intractable Diseases 074Program Number: 1684 Poster Board Number: D0319Presentation Time: 8:30 AM - 10:15 AMExpansion of corneal endothelial cells using biomimeticengineered substratesRachelle Palchesko 1, 3 , James L. Funderburgh 2, 3 , Adam W.Feinberg 1, 3 . 1 Biomedical Engineering, Carnegie Mellon University,Pittsburgh, PA; 2 Ophthalmology, University of Pittsburgh,Pittsburgh, PA; 3 Louis J Fox Center for Vision Restoration,University of Pittsburgh, Pittsburgh, PA.Purpose: Corneal endothelial cells (CECs) are non-proliferative invivo with minimal proliferation in vitro, making expansion of thesecells for therapeutic application difficult. Our previous work hasshown that culturing cells on a biomimetic substrate that mimics themechanical and biochemical properties of Descemet’s membraneenables the expansion of CECs >3000-fold compared to 139-fold ontissue culture polystyrene (TCPS). Here, we demonstrate that thebiomimetic substrate also maintains CEC phenotype and preventstransition to senescence or a fibroblast-like phenotype.Methods: CECs were isolated from bovine corneas and cultured onone of three different surfaces: the biomimetic substrate consisting ofcollagen type IV coated polydimethylsiloxane soft elastomer (COL4-PDMS) and two controls, TCPS and collagen type IV coated TCPS(COL4-TCPS). CECs were cultured for 8 passages andimmunofluorescently labeled at passages 1, 5, and 8 for fibronectin(FN), zonal occludins (ZO-1) and F-actin to analyze changes inextracellular matrix production, cell-cell coupling and polygonalmorphology. At these same time points qRT-PCR was used toquantify mRNA expression of COL4A2, COL8A1, and SLC4A4 asCEC markers and COL3A1 as the fibroblastic gene marker.Results: CECs cultured on the COL4-PDMS produced short,immature FN fibrils at all time points where as CECs on TCPS andCOL4-TCPS produced large fibrils at passages 5 and 8 similar tothose produced by fibroblasts. On COL4-PDMS CECs grew at higherdensity, had more continuous ZO-1 staining and maintained apolygonal morphology. Gene expression followed a similar patternwith fibroblast associated FN and COL3A1 genes higher on TCPSand COL4-TCPS and CEC associated COL8A1 and SLC4A4 genesmaintained at levels comparable to CECs in vivo on COL4-PDMS.Conclusions: We have demonstrated that a biomimetic substratewhich recapitulates the mechanical stiffness and collagen type IVcomposition of Descemet’s membrane significantly enhancesexpansion and maintains phenotypic stability of CECs in vitrocompared to TCPS and COL4-TCPS controls. Current efforts arefocused on extending this system to the expansion of human CECs toenhance its clinical relevance. The ability to expand CECs is crucialto obtain the cell numbers necessary for bioengineering a cornealendothelium suitable for implantation, a future goal of this researchproject.Commercial Relationships: Rachelle Palchesko, None; James L.Funderburgh, None; Adam W. Feinberg, Carnegie MellonUniversity (P)Support: This work was supported by the Ocular Tissue Engineeringand Regenerative Ophthalmology funding through the Louis J. FoxCenter for Vision Restoration, National Institutes of Health COREGrant P30 EY008098, EY09368 (to JLF), the Eye and EarFoundation of Pittsburgh, PA and an unrestricted Grant fromResearch to Prevent Blindness, New York, NYProgram Number: 1685 Poster Board Number: D0320Presentation Time: 8:30 AM - 10:15 AMMeganuclease Targeting HSV-1 Protects Against CornealEndothelitis ex-vivo and in-vivo©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.
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