Cornea - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaCommercial Relationships: Manuel Ramirez, None; EverardoHernandez-Quintela, NoneProgram Number: 5255 Poster Board Number: C0174Presentation Time: 2:45 PM - 4:30 PMInterclass Synergistic Effects of Small Leucine-RichProteoglycans in Regulating Collagen Fibrillogenesis DuringCorneal DevelopmentShoujun Chen 1 , Shukti Chakravarti 2 , David E. Birk 1 . 1 Department ofMolecular Pharmacology and Physiology, University of SouthFlorida, Tampa, FL; 2 Department of Medicine, Johns HopkinsUniversity, Baltimore, MD.Purpose: Small leucine-rich proteoglycans (SLRPs) are critical forthe regulation of stromal fibrillogenesis and corneal transparency.Class I and II SLRPs are enriched in the corneal stroma. These twofamilies bind to different sites on collagen fibrils. The purpose of thisstudy is to investigate cooperative interclass interactions in theregulation of fibrillogenesis in corneal stromal assembly.Methods: Compound Lum -/- /Bgn -/- mice were generated by crossbreedingthe single null mice. All experiments conformed to theARVO statement for the Use of Animals in Ophthalmic and VisionResearch. Stromal fibrillogenesis in this mouse model was analyzedusing in vivo confocal microscopy, immunohistochemistry, electronmicroscopy and molecular/biochemical approaches.Results: The phenotype of compound null mice was compared withwild type mice, Lum -/- mice, and Bgn -/- mice. The wild type mice andBgn -/- mice were phenotypically normal. Lum -/- mice exhibited cloudycorneas, while the opacity in corneas of compound Lum -/- /Bgn -/- micewas significantly greater. Both anterior and posterior light scatteringwere significantly increased in compound Lum -/- /Bgn -/- micecompared to Lum -/- mice as demonstrated by in vivo confocalmicroscopy. Disorganized thinner lamellae were observed usingelectron microscopy in the compound null mice compared to Lum -/-mice. These disorganized lamellae contain disorganized collagenfibrils with increased numbers of irregular, large diameter fibrils.These irregular collagen fibrils are heterogeneous in contour. Thedisorganized collagen fibrils also were observed at P10 when bothLum -/- mice and compound null mice have similar diameter collagenfibrils. Immunoreactivities for lumican or biglycan were not detectedin the corneas of compound Lum -/- /Bgn -/- mice as expected. Theimmuno-relativities of collagens I and V as well as other SLRPsincluding keratocan, fibromodulin and decorin were comparablebetween Lum -/- mice and compound null mice.Conclusions: The data indicate biglycan and lumican havesynergistic effects in regulating collagen fibrillogenesis duringcorneal stromal assembly. They demonstrate cooperative modulationof both linear and lateral fibril growth during early development andprovide the foundation for the regular collagen fibril organization incorneal stroma associated with transparency.Commercial Relationships: Shoujun Chen, None; ShuktiChakravarti, None; David E. Birk, NoneSupport: EY05129 and AR44745Program Number: 5256 Poster Board Number: C0175Presentation Time: 2:45 PM - 4:30 PMCollagen XII regulates collagen fibril packing, lamellarorganization, stromal architecture and corneal functionChinda Hemmavanh 1 , Edgar M. Espana 1, 2 , David E. Birk 1 .1 Molecular Pharmacology and Physiology, USF Morsani College ofMedicine, Tampa, FL; 2 Ophthalmology, University of South Florida,Tampa, FL.Purpose: Collagen XII is a fibril associated collagen (FACIT) thathas been hypothesized to regulate stromal organization andextracellular matrix interactions. Regulated fibril assembly, packing,and organization into orthogonal lamellae are vital for cornealfunction. The purpose of this study is to elucidate the role(s) ofcollagen XII in the regulation of fibril assembly and organization aswell as stromal structure which is required for corneal function.Methods: The role of collagen XII in the cornea was studied usingwild type and Col12a1-/- mice. Collagen XII expression patternswere analyzed using immuno-chemical, biochemical and molecularapproaches. Lamellae and fibril organization were analyzed usingtransmission electron microscopy in P30 corneas (n=3). Cornealthickness was measured at P30 and P60 using optical coherencetomography (n=6).Results: Collagen XII was homogeneously localized throughout thecorneal stroma in postnatal and mature corneas (P4-P90). Expressionfrom P4-P30 was relatively constant during this period with a slightdecrease in collagen XII expression at P90. Stromal organization wasdisrupted in the absence of collagen XII. Lamellar organization wasdisrupted in the Col12a1-/- compared to wild type corneas. Individuallamellae were disorganized with an increased density of fibrils in theCol12a1-/- corneas compared to controls. Mature Col12a1-/- corneaswere significantly thicker than the wild type controls. At P30, theCol12a1-/- corneal thickness was 132.2 ± 9.2μm (mean ± sd)compared to 101.4 ± 3.8μm for the wild type corneas. The P60thickness values were 117.4 ± 9.5μm and 93.8 ± 4.9μm for Col12a1-/- and wild type corneas respectively.Conclusions: Collagen XII is present in the corneal stroma duringcritical stages of postnatal development. The disruption of lamellarstructure and organization in Col12a1-/- corneas supports animportant regulatory role for collagen XII in higher orderextracellular matrix assembly, i.e., the organization of fibrils intowell ordered orthogonal lamellae. The data also support a role forcollagen XII in fibril packing with decreased interfibrillar spacingobserved in the collagen XII-null corneas. In addition, the dataindicate that the regulation of lamellar assembly has a role indetermination of corneal thickness. Ultimately collagen XII regulateslamellar organization, stromal integrity, and corneal function.Commercial Relationships: Chinda Hemmavanh, None; EdgarM. Espana, None; David E. Birk, NoneSupport: NEI Grant EY05129Program Number: 5257 Poster Board Number: C0176Presentation Time: 2:45 PM - 4:30 PMExtracellular matrix characterization of the acellular gammairradiatedcorneaJemin J. Chae 1 , Joshep S. Choi 1 , Walter Stark 2 , Jennifer Elisseeff 1 .1 Biomedical Engineering, Johns Hopkins University, Baltimore, MD;2 Ophthalmology, Johns Hopkins University, Baltimore, MD.Purpose: To assess and investigate the extracellular matrix (ECM) ofthe human acellular gamma-irradiated cornea (AGC) with respect tostructural, physical, mechanical and biological properties.Methods: The properties of the AGC and the equivalent fresh humancornea (FHC) were evaluated. The structure of corneas wereobserved by transmission electron microscopy (TEM) and lightmicroscopy. The physical and mechanical properties of ECM wereevaluated by differential scanning calorimetry (DSC), compressivemodulus testing and light transmission testing. To assess thebiological properties, a cell migration test with human keratocytesand a DNA content analysis were performed.Results: The structural properties of the AGC were generally similarto those of the FHC but the density of collagen fibers in the AGC waslower than that of the HFC and some cell debris was observed. Thesefindings were also supported by light microscopy. The AGC showeda higher light transmission rate, which corresponds with clarity,©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.