Cornea - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneadeficient mouse embryos, it will be of great relevance to investigatethe mechanism underlying Pnn’s impact on the alternative splicing ofHas2as. Finally, in situ hybridization analyses revealed the presenceof two splice variants of RP11-295G20.2, one of PNN-regulatedlncRNAs, in the nuclei of corneal epithelial cells, but not in thestromal cells of human cornea, suggesting their potential roles incorneal epithelial cells.Conclusions: The data strongly suggest PNN’s role in the alternativesplicing of a specific subset of lncRNAs that might have significantimpact on the corneal epithelium. (NIH Grant R01 EY007883, P30EY021721)Commercial Relationships: Jeong-Hoon Joo, None; Stephen P.Sugrue, NoneSupport: NIH Grant R01 EY007883, P30 EY021721Program Number: 2582 Poster Board Number: D0382Presentation Time: 2:45 PM - 4:30 PMLocalisation of Yap/Taz in corneal epithelia: a marker ofmechano-sensitivity and role in epithelial homeostasisChe J. Connon, Roanne R. Jones, James W. Foster. School ofChemistry, Food and Pharmacy, University of Reading, Reading,United Kingdom.Purpose: To investigate the cellular location of the nucleartranscription factor Yap/Taz in limbal and central corneal epithelialcells and relate this expression to changes in corneal stiffnesscentripetally across the ocular surface.Yap/Taz has recently been found to be an important regulator ofmechanotransduction. We believe that changes in substrate stiffnessoccur across the cornea and that a centripetal stiffness gradient drivesboth differentiation and migration of epithelial cells form the limbustowards the central cornea forming an important structuralcomponent of ocular surface homeostasis.Methods: The localisation of Yap/Taz, CK3, CK14 and ZO-1 acrossthe ocular surface of bovine corneas was examined byimmunohistochemistry. Limbal stem cells were isolated form freshbovine corneas and expanded upon type I collagen gels of differingstiffness for 14 days. The localisation of Yap/Taz, CK3, CK14 andZO-1 was examined by immunohistochemistry within these cornealconstructs. Furthermore, the transcription levels of Yap/Taz, CK3and ABCG2 were quantified by QPCR.Results: Across the healthy bovine cornea Yap/Taz waspredominately expressed cytoplasmically within the limbus, where asin central corneal epithelial cells Yap/Taz was retained within thenucleus. Isolated limbal epithelial cells expanded upon the morecompliant collagen gels showed significantly less gene expression ofYap/Taz, which was predominately cytoplasimic at the protein level,whereas more nuclear expression was seen within epithelial cellsexpanded upon the stiffer collagen gels. This corresponded with morecells expressing cytokeratin 3 and ZO-1 and less cytokeratin 14 andABCG2 at the gene and protein level.Conclusions: The nuclear to cytoplasmic expression ratio of Yap/Tazbetween limbal and central epithelial cells supports the notion of acentripetal stiffness gradient across the corneal surface. Thesuitability of YAP/Taz as a marker of mechanosensitivity (substratestiffness) in corneal epithelial cells was successfully demonstrated byuse of collagen gels as cell substrates with differing stiffness. Thepresence of a centripetal stiffness gradient across the cornea is likelyto underpin new directions in corneal wound healing and ourunderstanding of ocular surface homeostasis.Commercial Relationships: Che J. Connon, None; Roanne R.Jones, None; James W. Foster, NoneSupport: Medical Research Council G0900877/1Program Number: 2583 Poster Board Number: D0383Presentation Time: 2:45 PM - 4:30 PMVitamin D Receptor Knockout Affects Mouse Corneal TightJunctionsMitchell A. Watsky, Rodolfo A. Elizondo, Zhaohong Yin. Physiology,Univ of Tennessee Health Sci Ctr, Memphis, TN.Purpose: Vitamin D is typically known to be produced in the skinand activated through successive hydroxylation steps in the liver andkidneys. Our lab has recently determined that vitamin D can also beproduced in the cornea and can influence the barrier function of thecorneal epithelium. The purpose of this study was to determine ifproteins associated with corneal tight junctions are affected byvitamin D receptor (VDR) knockout and if the junctions themselvesare altered as observed using transmission electron microscopy(TEM).Methods: Wild type 2.5 month-old C57BL/6 mice along withheterozygous (+/-) and homozygous (-/-) VDR null mice (JacksonLabs) were used in this study. Total RNA was obtained from freshlyisolated mouse cornea epithelium. Real-time PCR was used toquantify mRNA levels of the tight junction-associated proteinsoccludin and ZO-1. 2ug total RNA was used for the subsequentsynthesis of cDNA using the ThermoScript RT-PCR system for firststrandsynthesis at 25°C 10min, 50°C 50min, and was inactivated byheating to 85°C for 5 min. Equal amounts of cDNA were applied forPCR amplification in quadruplicate at a final volume of 25 μl of 2XRT2 Real-Time SYBR Green master mix. Amplification wasperformed for 95°C 10min, followed by 40 cycles at 94°C 15 s and60°C 60 s. Quantitative values were obtained from the thresholdcycle value (Ct), which is the point where a significant increase offluorescence is first detected. GAPDH was used as an internal RNAcontrol, and each sample was normalized on the basis of its GAPDHgene content (ΔCt). Transmission electron microscopy (TEM) wasused to examine tight junction structures in the cornea epithelium andendothelium.Results: Occludin levels in -/- mice were significantly lower thanwild type and +/- mice. ZO-1 was significantly lower in -/- versuswild type mice. TEM demonstrated significant reductions inepithelial and endothelial tight junction structures in -/- corneasversus those from wild type mice.Conclusions: VDR ablation results in significant loss of tightjunction-associated proteins and tight junction structures in mousecorneas.Commercial Relationships: Mitchell A. Watsky, None; Rodolfo A.Elizondo, None; Zhaohong Yin, NoneSupport: NIH Grant EY021747Program Number: 2584 Poster Board Number: D0384Presentation Time: 2:45 PM - 4:30 PMPharmacological Analysis of Epidermal Growth Factor ReceptorLigands on Corneal Epithelial CellsBrian P. Ceresa 1, 2 , Joanne L. Peterson 2 . 1 Pharmacology andToxicology, University of Louisville, Louisville, KY; 2 Cell Biology,University of Oklahoma HSC, Oklahoma City, OK.Purpose: An active epidermal growth factor receptor (EGFR) is bothnecessary and sufficient for promoting the homeostasis of the cornealepithelium. However, stimulation of the EGFR with epidermalgrowth factor (EGF) has not been an effective strategy foraccelerating the repair of damaged corneal epithelium. Sincesynthetic EGFR ligands are not available, we have examined the roleof endogenous EGFR ligands in promoting corneal epithelial cellgrowth and migration. These findings will aid in developingalternative strategies for stimulating EGFR activity that will furtherour understanding of the progression and reversal of diseases of the©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.