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      Differential Distribution of Laminin N-Terminus α31 Across the Ocular Surface: Implications for Corneal Wound Repair

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          Abstract

          Purpose

          Laminin N-terminus (LaNt) α31 is a relatively unstudied protein derived from the laminin α3 gene but structurally similar to netrins. LaNt α31 has, to date, been investigated only in two-dimensional (2D) keratinocyte culture where it influences cell migration and adhesion, processes integral to wound repair. Here we investigated LaNt α31 distribution in ocular surface epithelium, during limbal stem cell activation, and corneal wound healing.

          Methods

          Human, mouse, and pig eyes, ex vivo limbal explant cultures, and alkali burn wounds were processed for immunohistochemistry with antibodies against LaNt α31 along with progenitor cell–associated proteins. LaNt α31 expression was induced via adenoviral transduction into primary epithelial cells isolated from limbal explants, and cell spreading and migration were analyzed using live imaging.

          Results

          LaNt α31 localized to the basal layer of the conjunctival, limbal, and corneal epithelial cells. However, staining was nonuniform with apparent subpopulation enrichment, and some suprabasal reactivity was also noted. This LaNt α31 distribution largely matched that of keratin 15, epidermal growth factor receptor, and transformation-related protein 63α (p63α), and displayed similar increases in expression in activated limbal explants. During active alkali burn wound repair, LaNt α31 displayed increased expression in limbal regions and loss of basal restriction within the cornea. Distribution returned to predominately basal cell restricted once the wounded epithelium matured. Cultured corneal epithelial cells expressing LaNt α31 displayed increased 2D area and reduced migration, suggesting a functional link between this protein and key wound repair activities.

          Conclusions

          These data place LaNt α31 in position to influence laminin-dependent processes including wound repair and stem cell activation.

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          Most cited references58

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          Laminins in basement membrane assembly

          The heterotrimeric laminins are a defining component of all basement membranes and self-assemble into a cell-associated network. The three short arms of the cross-shaped laminin molecule form the network nodes, with a strict requirement for one α, one β and one γ arm. The globular domain at the end of the long arm binds to cellular receptors, including integrins, α-dystroglycan, heparan sulfates and sulfated glycolipids. Collateral anchorage of the laminin network is provided by the proteoglycans perlecan and agrin. A second network is then formed by type IV collagen, which interacts with the laminin network through the heparan sulfate chains of perlecan and agrin and additional linkage by nidogen. This maturation of basement membranes becomes essential at later stages of embryo development.
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            The laminin family.

            Laminins are large molecular weight glycoproteins constituted by the assembly of three disulfide-linked polypeptides, the α, β and γ chains. The human genome encodes 11 genetically distinct laminin chains. Structurally, laminin chains differ by the number, size and organization of a few constitutive domains, endowing the various members of the laminin family with common and unique important functions. In particular, laminins are indispensable building blocks for cellular networks physically bridging the intracellular and extracellular compartments and relaying signals critical for cellular behavior, and for extracellular polymers determining the architecture and the physiology of basement membranes.
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              Characterization of putative stem cell phenotype in human limbal epithelia.

              This study evaluated proposed molecular markers related to stem cell (SC) properties with the intention of characterizing a putative SC phenotype in human limbal epithelia. Human corneal and limbal tissues were cut in the vertical and horizontal meridians for histology, transmission electron microscopy (TEM), and immunostaining. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization were used to evaluate gene expression. TEM showed that the limbal basal cells were small primitive cells. Immunostaining disclosed that p63, ABCG2 and integrin alpha9 were primarily expressed by the basal epithelial cells of limbus. Antibodies against integrin beta1, epidermal growth factor receptor (EGFR), K19, enolase-alpha, and CD71 stained the basal cells of the limbus more brightly than the suprabasal epithelia. Integrin alpha6, nestin, E-cadherin and connexin 43 did not stain the limbal basal cells, but the suprabasal epithelia of the cornea and limbus showed strong immunoreactivity. K3 and involucrin stained only corneal and limbal superficial cells. RT-PCR showed higher levels of p63, ABCG2 and integrin alpha9 mRNA, but lower levels of K3, K12 and connexin 43 expressed in the limbal epithelia than the corneal epithelia. In situ hybridization showed that p63 transcripts were located in basal layer of the limbal epithelium. This work suggests that the basal epithelial cells of the limbus are p63, ABCG2 and integrin alpha9 positive, and nestin, E-cadherin, connexin 43, involucrin, K3, and K12 negative, with relatively higher expression of integrin beta1, EGFR, K19, and enolase-alpha. This putative SC phenotype may facilitate the identification and isolation of limbal epithelial SCs.
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                Author and article information

                Journal
                Invest Ophthalmol Vis Sci
                Invest. Ophthalmol. Vis. Sci
                iovs
                Invest Ophthalmol Vis Sci
                IOVS
                Investigative Ophthalmology & Visual Science
                The Association for Research in Vision and Ophthalmology
                0146-0404
                1552-5783
                August 2018
                : 59
                : 10
                : 4082-4093
                Affiliations
                [1]Department of Eye and Vision Science, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, United Kingdom
                Author notes
                Correspondence: Kevin J. Hamill, Department of Eye and Vision Science, Institute of Ageing and Chronic Disease, William Henry Duncan Building, University of Liverpool, L7 8TX, 6 West Derby Street, Liverpool, UK; khamill@ 123456liverpool.ac.uk .

                VB and LDT contributed equally to the work presented here and should therefore be regarded as equivalent authors.

                Article
                iovs-59-10-06 IOVS-18-24037R2
                10.1167/iovs.18-24037
                6735649
                30098195
                23eb385c-597d-45ac-abdd-a8518a285bcd
                Copyright 2018 The Authors

                This work is licensed under a Creative Commons Attribution 4.0 International License.

                History
                : 5 February 2018
                : 28 June 2018
                Categories
                Cornea

                cornea,ocular surface epithelium,corneal wound healing,basement membrane,laminin

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