Cornea - ARVO

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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaan elongated and interconnected fibroblastic morphology, and asignificant increase in cellular light scattering was measured. Thisstromal haze gradually decreased as wound healing progressed.Conclusions: Overall, this modified system allows high resolution 3-D image stacks from the full thickness rabbit cornea to be obtainedduring in vivo wound healing. These datasets can be used forinteractive visualization of corneal cell layers, measurement of sublayerthickness, assessment of cell morphology and connectivity, andestimation of stromal backscatter (haze) during wound healing.Commercial Relationships: Matthew Petroll, None; Daniela B.Hagenasr, None; H D. Cavanagh, Menicon Ltd (C); Danielle M.Robertson, NoneSupport: NIH Grants EY013322 and EY020799, and Research toPrevent Blindness, Inc.Program Number: 3223Presentation Time: 12:30 PM - 12:45 PMOptical coherence tomography (OCT) combined withvideokeratography to detect ‘early’ keratoconus - a new“pachymetry/asymmetry” index to quantify disease severityYaron S. Rabinowitz, Yelena Bykhovskaya, Xiaohui Li, Ana Laura C.Canedo. Cornea Genetic Eye Institute, Beverly Hills, CA.Purpose: To develop parameters using a combination of opticalcoherence tomography (OCT) and videokeratography to ‘early’detect keratoconus.Setting: Study was performed at the Cornea Genetic Eye Institute inBeverly Hills, CA.Methods: We studied the following groups: 180 normal eyes, 46 eyewith moderate keratoconus, 54 eyes with early keratoconus, 7 eyeswith ‘forme fruste’ keratoconus and 16 keratoconus ‘suspects’. Weperformed videokeratography, wavefront analysis and measured OCTindices to determine which combination was the most sensitive forseparating all study groups.Results: A combination of videokeratography and OCT indices (I-Svalue and Minimum pachymetry) was statistically the mostsignificant in separating keratoconus groups from normals (P
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaMethods: Expression of the TRA-1-60 antigen was investigated incorneo-limbal specimens from 10 human donor eyes using light andelectron microscopic immunohistochemistry. TRA-1-60 mRNAexpression was analyzed after laser capture microdissection of basallimbal epithelial cell clusters and limbal stromal cells, respectively,and pre-amplification of RNA by real-time PCR. In addition,antibodies against putative stem/progenitor markers, includingABCG2, p63alpha, cytokeratin 15, N-Cadherin, Sox-9, and Oct-4, aswell as against laminin-1, Melan A, CD11c, and CD45 were used indouble labelling experiments.Results: The TRA-1-60 epitope was abundantly expressed on thesurface of a subset of mesenchymal cells in the anterior limbal stromadisplaying a well-defined demarcation to the peripheral cornealstroma. The TRA-1-60 positive cells cumulated adjacent to limbalbasal epithelial cell clusters expressing putative stem/progenitormarkers and established intimate contact with epithelial cells bynumerous cell processes. In addition, few TRA-1-60 positive cellswere found to be integrated within the basal epithelial layer overlyingthe epithelial basement membrane. Dual staining showed that allTRA-1-60 positive cells co-expressed vimentin, but were negative forMelan A, CD11c, and CD45 excluding their nature as intraepithelialmelanocytes, dendritic cells or lymphocytes. Moreover, TRA-1-60staining was also associated with perivascular cells of anteriormostlimbal blood vessels. Real-time PCR confirmed differentialexpression of TRA-1-60 in limbal stromal cells compared to cornealstromal cells.Conclusions: The unequivocal presence of pluripotent cells withinsubepithelial stroma, blood vessel walls, and basal epithelium at thehuman limbus provides new insights into the constitution of thelimbal niche and its interaction with limbal epithelial stem/progenitorcells. These cells may have a great potential in corneal regenerationand wound healing.Commercial Relationships: Friedrich E. Kruse, None; NareshPolisetti, None; Johannes Menzel-Severing, None; UrsulaSchlotzer-Schrehardt, NoneProgram Number: 3226Presentation Time: 11:30 AM - 11:45 AMDonor MSCs expressing MHC class II molecule trigger immuneresponses in an animal model of lacrimal gland chronic graftversus host diseaseYoko Ogawa, Shigeto Shimmura, Saori Yaguchi, Takaaki Inaba,Kazuo Tsubota. Department of Ophthalmology, Keio Univ School ofMedicine, Shinjuku-Ku, Japan.Purpose: To examine using a cGVHD mouse model whether donorMSCs expressing MHC class II have a potential to trigger lacrimalgland cGVHD.Methods: We chose a MHC-compatible, minor antigen (miHA)-incompatible model of cGVHD involving bone marrowtransplantation (WBMT) with 8 week-old donor B10.D2 (H-2d) miceand recipient BALB/c mice (H-2d). We first modified the cGVHDmodel by co-transplanting prospectively isolated HSCs withprospectively isolated MSCs. This model closely mimics clinicalfindings of patients suffering from cGVHD. We examined lacrimalgland inflammation and fibrosis, expression of MHC class II onMSCs, T cell subsets and cytokine production in peripheral bloodafter the co-transplantation.Results: We found that serum levels of IL-6 increased in recipientmice transplanted with mismatched MSCs starting at 3 weeks aftertransplantation. This coincided with the appearance of lacrimal glandinflammation and fibrosis and MSC-derived cells in the peripheralblood of recipients starting at 3 weeks after transplantation andgradually increasing up to 7 weeks. Interestingly, these MSC-derivedcells in the peripheral blood expressed MHC class IIantigens.Flowcytometry revealed that both MSCs and T cells frommismatched MSC-transplanted recipients produce IL-6. ProliferatedT cells are predominantly recipient derived CD4+ T cells. MHC classII molecule was expressed in a subset of MSC cells, but thefrequency was significantly increased after co-culture with recipientT cells.Conclusions: These data suggest that mismatched MSCs and host Tcells stimulate each other via the MHC class II molecule. Our resultsshow that donor MSCs expressing MJHC class II molecule triggerimmune responses in cGVHD related dry eye, challenging currentparadigms on the pathogenesis of the disease.Commercial Relationships: Yoko Ogawa, None; ShigetoShimmura, None; Saori Yaguchi, None; Takaaki Inaba, None;Kazuo Tsubota, AcuFocus, Inc (C), Allergan (F), Bausch LombSurgical (C), Functional visual acuity meter (P), JiNS (P), Kissei (F),Kowa (F), Santen, Inc. (F), Otsuka (F), Pfizer (C), Thea (C), EchoDenki (P), Nidek (F), Ophtecs (F), Wakasa Seikatsu (F), CEPTCompany (P)Support: Japanese Ministry of Education, Science, Sports andCulture# 23592590Program Number: 3227Presentation Time: 11:45 AM - 12:00 PMIntegral Analysis of Gene Signatures and MicroRNA Expressionof Cultured Human Corneal Endothelial Cells in Relation toTheir Functions, Cell Senescence, Epithelial-MesenchymalTransition, and FibrosisKazuko Asada 1 , Munetoyo Toda 1 , Michio Hagiya 1 , Kana Nakata 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, Kyotanabe, Japan.Purpose: Premature cell senescence in cultured human cornealendothelial cells (HCECs) is partly due to epithelial-mesenchymaltransition (EMT), a phenotypic switch in which HCECs undergophenotypic changes resulting in dampened cell propagation. In thisstudy, we investigated and compared HCEC gene-signature featuresbetween HCECs cultured with or without EMT-like phase transitionto understand the molecular aspects underlying the difficulty inpropagating HCECs in culture.Methods: Gene expression signatures of cultured HCECs with nosign of EMT and those with EMT-like phenotypic changes werecompared by use of polymerase chain reaction (PCR) array assay.Due to increasing evidence indicating that microRNA (miR)regulates gene functions, we also integrally analyzed miR expressionin corneas from distinct donors and cultured HCECs without EMTliketransition.Results: Fresh donor HCECs elicited completely distinct genesignatures from those of corneal epithelial cells from the same tissues[correlation coefficient (CC), 0.58~0.62]. Of interest, miR profileswere analogous with those from epithelium (CC, 0.95). Irrespectiveof the presence of EMT-like transition during culture, mRNA-basedgene signatures similar to those of fresh tissue were revealed (CC,0.7~0.8), while those of miR were far distinct (CC, 0.25~0.50). Thisimplicates the crucial roles of miR in regulating the functional genesof HCECs. The expression profile of miR varied dramaticallybetween donors 20 and 60 years of age. In the integral PCR arrayassay of gene signatures relating to EMT-like transition, fibrosisrevealed a significant decrease in the expression of distinct genes.Moreover, many senescence- and cell-cycle-related genes weredownregulated in the cultured HCECs with EMT-like transition.Conclusions: Gene-signature features of HCECs and corneal©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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