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Loss of corneal epithelial heparan sulfate leads to corneal degeneration and impaired wound healing.

ABSTRACT: PURPOSE:Heparan sulfate (HS) is a highly modified glycosaminoglycan (GAG) bound to a core protein to form heparan sulfate proteoglycans (HSPGs) that are vital in many cellular processes ranging from development to adult physiology, as well as in disease, through interactions with various protein ligands. This study aimed to elucidate the role of HS in corneal epithelial homeostasis and wound healing. METHODS:An inducible quadruple transgenic mouse model was generated to excise Ext1 and Ndst1, which encode the critical HS chain elongation enzyme and N-deacetylase/N-sulfotransferase, respectively, in keratin 14-positive cells upon doxycycline induction. RESULTS:EXT(?/?CEpi) mice (deletion of Ext1 in corneal epithelium) induced at P20 presented progressive thinning of the corneal epithelium with a significant loss in the number of epithelial layers by P55. EXT(?/?CEpi) mice presented tight junction disruption, loss of cell-basement membrane adhesion complexes, and impaired wound healing. Interestingly, EXT(?/?CEpi) and NDST(?/?CEpi) mice presented an increase in cell proliferation, which was assayed by both Ki67 staining and 5-ethynyl-2'-deoxyuridine (EdU) incorporation. Moreover, EXT(?/?CEpi) mice presented compromised epithelial stratification 7 days after a debridement wound. The conditional knockout of HS from keratocytes using the keratocan promoter led to no corneal abnormalities or any disruption in wound healing. CONCLUSIONS:Corneal epithelial cells require HS for maintaining corneal homeostasis, and the loss of epithelial HS leads to both impaired wound healing and impaired corneal stratification.

SUBMITTER: Coulson-Thomas VJ

PROVIDER: S-EPMC4432551 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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Loss of corneal epithelial heparan sulfate leads to corneal degeneration and impaired wound healing.

Coulson-Thomas Vivien Jane VJ Chang Shao-Hsuan SH Yeh Lung-Kun LK Coulson-Thomas Yvette May YM Yamaguchi Yu Y Esko Jeffrey J Liu Chia-Yang CY Kao Winston W

Investigative ophthalmology & visual science 20150501 5

<h4>Purpose</h4>Heparan sulfate (HS) is a highly modified glycosaminoglycan (GAG) bound to a core protein to form heparan sulfate proteoglycans (HSPGs) that are vital in many cellular processes ranging from development to adult physiology, as well as in disease, through interactions with various protein ligands. This study aimed to elucidate the role of HS in corneal epithelial homeostasis and wound healing.<h4>Methods</h4>An inducible quadruple transgenic mouse model was generated to excise Ext ...[more]

PMID: 26024086

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Project description:Following injury, corneal stromal keratocytes transform into repair-phenotype of activated stromal fibroblasts (SFs) and participate in wound repair. Simultaneously, ongoing bi-directional communications between corneal stromal-epithelial cells also play a vital role in mediating the process of wound healing. Factors produced by stromal cells are known to induce proliferation, differentiation, and motility of corneal epithelial cells, which are also subsequently the main processes that occur during wound healing. In this context, the present study aims to investigate the effect of SFs conditioned medium (SFCM) on corneal epithelial cell function along with substance P (SP). Antibody microarrays were employed to profile differentially expressed cell surface markers and cytokines in the presence of SFCM and SP. Antibody microarray data revealed enhanced expression of the ITGB1 in corneal epithelial cells following stimulation with SP whereas SFCM induced abundant expression of IL-8, ITGB1, PD1L1, PECA1, IL-15, BDNF, ICAM1, CD8A, CD44 and NTF4. All these proteins have either direct or indirect roles in epithelial cell growth, movement and adhesion related signaling cascades during tissue regeneration. We also observed activation of MAPK signaling pathway along with increased expression of focal adhesion kinase (FAK), paxillin, vimentin, ?-catenin and vasodilator-stimulated phosphoprotein (VASP) phosphorylation. Additionally, epithelial-to-mesenchymal transition (EMT) regulating transcription factors Slug and ZEB1 expression were enhanced in the presence of SFCM. SP enriched the expression of integrin subunits ?4, ?5, ?V, ?1 and ?3 whereas SFCM increased ?4, ?5, ?V, ?1 and ?5 integrin subunits. We also observed increased expression of Serpin E1 following SP and SFCM treatment. Wound healing scratch assay revealed enhanced migration of epithelial cells following the addition of SFCM. Taken together, we conclude that SFCM-mediated sustained activation of ZEB1, Slug in combination with upregulated migration-associated integrins and ERK (Extracellular signal-regulated kinase)-FAK-paxillin axis, may lead to induce type 2 EMT-like changes during corneal epithelial wound healing.

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Loss of corneal epithelial heparan sulfate leads to corneal degeneration and impaired wound healing.

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Loss of corneal epithelial heparan sulfate leads to corneal degeneration and impaired wound healing.

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