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Obi Onochie, Vickery E Trinkaus-Randall; Hypoxia Affects Cell Migration and Epithelial Wound Healing by Altering Basal Lamina Proteins and Focal Adhesion Turnover. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):706.
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© ARVO (1962-2015); The Authors (2016-present)
Corneal hypoxia can result from conditions such as diabetes and corneal infections. Our previous data indicate that hypoxia leads to delayed wound healing compared to controls. The aim of this study is to further examine basal lamina compositional changes and to determine how these alterations affect focal adhesions and other proteins involved in migration.
An ex vivo corneal organ culture model was employed to examine cell migration and wound healing. Injuries were made using a 3 millimeter trephine and corneas were incubated in normoxia or hypoxia and fixed at different timepoints for immunohistochemistry (IHC). An in vitro scratch wound model was employed with human corneal limbal epithelial (HCLE) cells. Cultures were exposed to normoxia or hypoxia and fixed for IHC or probed for western blot analysis.
Wounded corneas exposed to hypoxia displayed decreased punctate staining of syndecan-1 along the basal lamina and throughout the epithelium compared to normoxic corneas. Fibronectin expression decreased along the basal lamina and anterior stroma, but remained unchanged in basal cells compared to normoxic corneas. In hypoxic corneas, vinculin was diffusely expressed throughout the epithelium rather than concentrating within the cytoplasm of basal cells. Also, there was a loss of apical vinculin expression. Cytoplasmic punctate expression of vinculin Y1065 decreased while expression remained along the basal lamina. Paxillin expression within basal cells and along the apical surface of the epithelium was reduced under hypoxia. Paxillin Y118 was continuous along the basal cell membrane, but lacked the punctate staining seen in normoxic corneas. Furthermore, paxillin Y118 decreased throughout the epithelium after exposure to hypoxia. While western blot analysis of unwounded HCLEs shows no change in vinculin after 24 hours of hypoxia, vinculin Y1065 was enhanced three fold.
Using the ex vivo model we can investigate impaired wound healing in various pathologies involving corneal hypoxia. Determining alterations in basal lamina composition and properties and characterizing the changes in regulation and dynamics of focal adhesion proteins, will allow for the development of treatment modalities.
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