Cornea - ARVO

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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneanerve and CD45+ cells were quantified. Pull down experiments usingGAL3-conjugated agarose beads were performed to determine ifTrkA binds to GAL3.Results: Robust neurite outgrowth was seen in OTG culturesincubated with NGF, as compared to cultures in media alone(p
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneamustard (NM) to determine whether hydroxamate compoundsfacilitate recovery. NM is a vesicating agent that causes separation atthe epithelial-stromal junction, and can lead to sloughing of theepithelium 1-2 days after exposure. Separation of the cell layersinvolves, in part, cleavage of hemidesmosomal collagen XVII byADAM17. Hydroxamates inhibit ADAM17 via binding to theenzyme’s catalytic zinc. Our goal was to evaluate two hydroxamatesfor their ability to inhibit ADAM17 and to explore other mechanismsfor how they favor epithelial-stromal integrity after injury.Methods: Rabbit corneas in organ culture were exposed to 100 nmolNM (in 10 ul) for 2 hr. Corneas were then washed, medium waschanged, and 3 nmol of each hydroxamate (in 20 ul) was applied toNM-exposed corneas 4 times over the course of 22 hrs. Thehydroxamate compounds used as therapies were olvanil OH(NDH4409) and retro olvanil 8 (NDH4417).Results: Without hydroxamate treatment, ADAM17 was activated inthe cornea immediately upon exposure to nitrogen mustard (NM).Activation persisted for a minimum of 24 hr post exposure,correlating with epithelial-stromal separation. With the application ofeither hydroxamate, epithelial sloughing was reduced at 24 hr postexposure, and the overall histology of the basement membrane zonewas greatly improved. Surprisingly, the improvement of histology didnot correlate with the ability of the hydroxamates to inhibitADAM17. NDH4417 was a much better inhibitor than NDH 4409 inactivity assays. Since matrix metalloproteinases (MMPs) also containcatalytic Zn ions, both NDH4417 and NDH4409 were tested for theirability to reduce MMP9 levels, and both were found to be veryeffective. Next, to investigate an upstream step in MMP activation,the expression of the MMP inducer, EMMPRIN, was evaluated.EMMPRIN was found to be upregulated by mustard exposure. BothNDH4409 and NDH4417 treatment after NM exposure reducedEMMPRIN expression levels to values lower than unexposedcontrols, despite that EMMPRIN is not known to contain a zincmoiety.Conclusions: The hydroxamates employed not only reduce theactivity of ADAM17 and MMPs, but they also lower MMP levels byattenuating expression of the MMP inducer, EMMPRIN. Themechanism behind this latter activity is as yet unknown.Commercial Relationships: Marion K. Gordon, None; Andrea D.Rodrigues, None; Rita A. Hahn, None; Donald R. Gerecke, None;Kathy K. Svoboda, None; Ned D. Heindel, NoneSupport: NIH NEI R01 EY0090576; NIH NIAMSD U54AR055073; NIH NIEHS P30 ES005022Program Number: 3911 Poster Board Number: D0155Presentation Time: 2:45 PM - 4:30 PMAn intracellular degradation pathway implicated in cornealscarringAudrey Bernstein, Lingyan Wang, Stephanie R. Gillespie.Ophthalmology, Mount Sinai School of Medicine, New York, NY.Purpose: Scarring in the cornea can lead to vision loss. Corneal hazeand stromal fibrosis is the result of the persistence of myofibroblasts(Mfs), which excessively contract tissue and secrete fibroticextracellular matrix. We recently reported that corneal Mfs arecharacterized by cell surface accumulation of integrin αvβ5 resultingfrom decreased integrin degradation. An agnostic screening approach(RNA-seq) was used to identify possible molecular mechanisms.Methods: Methods: Primary human and porcine corneal fibroblastswere cultured. Gene expression was quantified by RNA-seq, alphasmoothmuscle actin (α-SMA) and integrin expression by Westernblotting and RT-PCR, and protein complex formation by co-IP.Transfection of cDNA was by Nucleofection. Mfs were detected withantibody to α-SMA. The EDA-type of fibronectin (FN-EDA) wasimaged by confocal microscopy and quantified with Leica software.A recycling assay quantified recycling of integrins. Porcine corneas(wounded by keratectomy and control) were cultured for 2 weeksprior to histological examination. USP10 expression was quantifiedwith Image J software.Results: RNA-seq screening revealed that Mfs have increased geneexpression of a subset of de-ubiquitinases (Dubs). Dubs removeubiquitin from proteins, saving them from degradation therebystabilizing protein levels. We found that the Dub, ubiquitin specificpeptidase 10 (USP10), controls integrin αvβ5 protein levels and isincreased in Mfs. This fits our recent finding that Mf differentiationoccurs when integrin αvβ5 ubiquitination and degradation is reduced.Furthermore, over-expression of USP10 cDNA led to a 3-foldincrease in integrin αvβ5 protein expression (with no increase inRNA expression) and a 70% increase in recycling of integrin αvβ5 tothe cell surface (because of reduced degradation). USP10 overexpressionalso induced fibrotic proteins, a 2-fold increase in α-SMA,a 4.4-fold increase in FN-EDA with organization of extracellular FN-EDA when USP10 is over-expressed but not in control. Co-IPconfirmed that USP10 is in a complex with integrin αvβ5. Finally,keratectomy generated stromal Mfs with a 6.8-fold increase in USP10expression.Conclusions: Mfs have increased USP10 expression andexperimental USP10 over-expression induced fibrotic markers.Cellular degradation systems and USP10 in particular may be a novelanti-scarring target.Commercial Relationships: Audrey Bernstein, None; LingyanWang, None; Stephanie R. Gillespie, NoneSupport: This research was supported by NIH-NEI Grant EY09414,NEI Core Grant P30-EY01867, and a Research to Prevent Blindnessgrant. The authors acknowledge use of human tissues provided by theNational Disease Research Interchange (NDRI), with support fromNIH grant 5U42RR006042. Microscopy was performed at theMSSM-Microscopy Shared Resource Facility, supported withfunding from NIH-NCI shared resources grant (5R24CA095823-04),a NSF Major Research Instrumentation grant (DBI-9724504), and aNIH shared instrumentation grant (1S10RR09145-01).Program Number: 3912 Poster Board Number: D0156Presentation Time: 2:45 PM - 4:30 PMMast Cells and the Inflammatory Response to Corneal EpithelialAbrasionAlan R. Burns 1, 2 , Qiong Liu 2 , Zhijie Li 2 , Clifton W. Smith 2 . 1 Collegeof Optometry, University Eye Institute, Houston, TX;2 Pediatrics/Leukocyte Biology, Baylor College of Medicine,Houston, TX.Purpose: Inflammation is beneficial to corneal epithelial and nerveregeneration following abrasion injury. Numerous studies havedocumented a regulatory role for mast cells in inflammation, tissueregeneration and wound healing. The mast cell contribution toinflammation and wound healing after epithelial abrasion isunknown.Methods: Anesthetized adult wildtype C57BL/6 mice or mast celldeficient mice (c-kit-/-) received a 2.0 mm diameter central cornealepithelial abrasion using a golf-club spud. To inhibit mast celldegranulation, wildtype mice were pretreated with cromoglycate(orally, topically or intraperitoneally) or ketotifen (topically). For allmice, topical application of fluorescein was used to evaluate the rateof epithelial wound closure. Excised corneal whole mounts wereprepared for immunofluorescence microscopy and used to evaluatechanges in limbal vessel diameter, numbers of extravasated plateletsand neutrophils and numbers of dividing epithelial cells.Results: Mast cells were abundant near limbal vessels in wildtype©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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