Cornea - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaPresentation Time: 3:15 PM - 3:30 PMIdentification of New markers of Human Corneal EndothelialCellsJodhbir S. Mehta 1 , Adrian Cheong 2 , Gary S. Peh 1 , William Sun 2 .1 Cornea Refractive Tissue Engineering, SNEC / SERI, Singapore,Singapore; 2 Experimental Therapeutic Unit, A Star, Singapore,Singapore.Purpose: There are no specific markers of human corneal endothelialcells (HCEC). Currently, the identification of HCEC in culture reliesmainly on expression of pump or tight junction markers.The aim ofthis study is to describe new HCEC markers.Methods: Isolated human corneal DM-HCEC and remaining stromafrom corneal donors were homogenized individually for RNAsequencing. RNA-seq libraries were prepared using AB protocol fornon-barcoded libraries and SOLiD fragment library for barcodedlibrary. The results were processed using ABI Bioscope. Geneexpression was measured by counting the number of reads mappinguniquely to both strands of each gene footprint. Results were verifiedby quantitative PCR, BD lyoplate screening, immunofluorescenceand flow cytometry using cultivated primary HCEC isolated andgrown to the third passage.Results: RNA-seq showed 5 genes that were over expressed in theHCEC-DM compared to stromal fibroblasts, GPC4, CNTN6,SLC9A7, PVRL3, and SLC4A4. The resulting over-expression ofthese genes was confirmed on comparing qPCR data from culturedHCEC and stromal fibroblasts. BD lyoplate identified CD104 andCD200 as potential markers. On immuno-histochemistry anti-GPC4,anti-CD200 and anti-SLC4A cell-surface antibodies clearly stainedthe the endothelial layer specifically. In a population of pre-CMFDAlabelled stromal fibroblasts and HCEC, mixed in a 1:1ratiofluorescent-activated cell sorting (FACS) showed a 96% recovery ofHCEC when sorted with anti-GPC4 and a 79.8% with anti-CD200.Conclusions: By RNA sequencing verified on qPCR andimmunohistochemistry we have identified two novel cell surfaceantigens on human corneal endothelial cells. These markers may beused to aid cell purification during harvesting or in the identificationof cultured HCEC to ensure quality control of cultured cells.Commercial Relationships: Jodhbir S. Mehta, None; AdrianCheong, None; Gary S. Peh, Singapore Eye Research Institute (P);William Sun, NoneSupport: NMRC TCRP 2011Program Number: 2199Presentation Time: 3:30 PM - 3:45 PMTight Junction Transmembrane Protein Claudin SubtypeExpression and Distribution in Human Corneal EndotheliumEmi Inagaki, Shin Hatou, Satoru Yoshida, Hideyuki Miyashita, KazuoTsubota, Shigeto Shimmura. Ophthalmology, Keio University,Tokyo, Japan.Purpose: The primary function of corneal endothelium is to maintaincorneal transparency by regulating corneal hydration and nutritionmodulated by barrier and metabolic pump function. However, themolecular mechanism of its barrier function is still relativelyunknown. Claudins, recently identified main components of tightjunctions,are a family of four-transmembrane-spanning proteins. Todate, 24 subtype of claudins have been identified in human.Combination of claudin subtypes may contribute not only to thetightness of tight junction strands but also ion-selective channels. Inthis study, we investigated the expression and pattern of claudins inin vivo human cornea.Methods: The experiments in this paper used remained corneal tissuesupplied from USA eye bank after the central buttons were used forcorneal transplantation. We stripped corneal endothelium withDescemet membrane and corneal epithelium from corneal stroma.Reverse transcription-polymerase (RT-PCR) was performed toevaluate that subtypes of claudins expressed in corneal endothelium,stroma and endothelium. Then, immunohistochemistry wasperformed for claudins positively expressed in RT-PCR, to confirmwhether they would express lateral side of corneal endothelium.Results: Transcripts for claudin-1, -2, -3, -4,-7, -10b, -11,-12,-14, -15, -22, -23, and -24 were identified in human corneal endothelium inRT-PCR. Claudin-1, -2, -3, -4, -7 , -11, -12, -14, -15, -22, -23, and -24 expression in corneal endothelium was common to cornealepithelium, and claudin-1, -2,-3,-4, -7, -11,-12,-14, -15, -22,-23,and -24 was common to corneal stroma. Among the claudin subtypesexpressed in corneal endothelium, immunohistochemistry revealedthe expression of claudin-1,-2, -4, -7, -10 antibodies showed bandsthat correspond to the junctional complex. Claudin-11 was alsoexpressed in corneal endothelium, however, the expression was notcontinuous but in a dotlike pattern along cell junctions.Conclusions: Claudin-1, -2, -4,-7, -10b and -11 are expressed incorneal endothelium. This combination of claudin subtype maycontribute to the uniqueness of barrier integrity and ion sensitivity incorneal endothelium.Commercial Relationships: Emi Inagaki, None; Shin Hatou,None; Satoru Yoshida, None; Hideyuki Miyashita, None; KazuoTsubota, AcuFocus, Inc (C), Allergan (F), Bausch Lomb Surgical(C), Functional visual acuity meter (P), JiNS (P), Kissei (F), Kowa(F), Santen, Inc. (F), Otsuka (F), Pfizer (C), Thea (C), Echo Denki(P), Nidek (F), Ophtecs (F), Wakasa Seikatsu (F), CEPT Company(P); Shigeto Shimmura, NoneProgram Number: 2200Presentation Time: 3:45 PM - 4:00 PMCorneal endothelial cells provide evidence of accelerated cellularsenescence associated with HIV infection: a case-control studySophia Pathai 1, 2 , Stephen D. Lawn 3, 2 , Paul G. Shiels 5 , Helen A.Weiss 4 , Colin Cook 6 , Robin Wood 2 , Clare E. Gilbert 1 . 1 InternationalCentre for Eye Health, London School of Hygiene & TropicalMedicine, London, United Kingdom; 2 Desmond Tutu HIV Centre,University of Cape Town, Cape Town, South Africa; 3 Dept ofClinical Research, London School of Hygiene & Tropical Medicine,London, United Kingdom; 4 MRC Tropical Epidemiology Group,London School of Hygiene & Tropical Medicine, London, UnitedKingdom; 5 Dept of Epigenetics, Institute of Cancer Sciences,University of Glasgow, Glasgow, United Kingdom; 6 Dept ofOphthalmology, University of Cape Town, Cape Town, South Africa.Purpose: Cellular senescence may be a key factor in HIV-relatedpremature biological aging. We assessed features of the cornealendothelium that are known to be associated with biological aging,and cellular senescence markers in HIV-infected adults.Methods: Case-control study of 242 HIV-infected adults and 249matched controls. Using specular microscopy, the cornealendothelium was assessed for features of aging (low endothelial celldensity [ECD], high variation in cell size, and low hexagonalityindex). Data were analysed by multivariable regression. CDKN2A (acell senescence mediator) and 8-hydroxy-2′-deoxyguanosine (anoxidative DNA damage marker) were measured in peripheral bloodleukocytes.Results: The median age of both groups was 40 years. Among HIVinfectedadults, 88% were receiving antiretroviral therapy (ART);their median CD4 count was 468 cells/μL. HIV infection wasassociated with increased odds of variation in cell size (OR=1.67;95%CI: 1.00-2.78, p=0.04). Among HIV-infected participants, lowECD was independently associated with current CD4 count