title: Perlecan-deficient mutation impairs corneal epithelial structure.
abstract: OBJECTIVE
  To elucidate the role of perlecan (Hspg2), a large multidomain heparan sulfate proteoglycan expressed in the basement membrane, in the structure of the corneal epithelium.
  
  
  METHODS
  A previously developed perlecan-deficient (Hspg2⁻/⁻-Tg) mouse model was used. Histologic analysis of their corneas was performed by light and transmission electron microscopy. The localization of perlecan in the corneas of wild-type (WT) mice and Hspg2⁻/⁻-Tg mice was examined by immunohistochemistry. The effects of perlecan deficiency on corneal epithelial structure was analyzed with respect to the expression of corneal epithelial proliferation and differentiation markers, such as Ki67, cytokeratin12 (K12), connexin43 (Cx43), Notch1, and Pax6 by immunohistochemistry and real-time polymerase chain reaction (PCR).
  
  
  RESULTS
  The Hspg2⁻/⁻-Tg mice had microphthalmos and a thinner corneal epithelium compared with that of the WT mice. Perlecan was localized in the corneal epithelial basement membrane in the WT mice, but not in the Hspg2⁻/⁻-Tg mice. The Hspg2⁻/⁻-Tg corneal epithelium exhibited thinner wing cell layers and a decreased number of Ki67-positive cells, but no dead cells, compared with the WT corneal epithelium. Immunohistochemistry and real-time PCR analysis revealed a significantly decreased expression of corneal epithelial differentiation markers such as K12, Cx43, Notch1, and Pax6 in Hspg2⁻/⁻-Tg mice, compared with those of the WT mice.
  
  
  CONCLUSIONS
  The findings of this study highlight a strong correlation between the presence of perlecan in the basement membrane and the structure of corneal epithelium and that the perlecan-deficient mutation impairs corneal epithelial structure.
authors.affiliation: Department of Ophthalmology, Juntendo University School of Medicine, Tokyo, Japan.
authors.forename: Takenori
authors.initials: T
authors.lastname: Inomata
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Ebihara
authors.forename: Toshinari
authors.initials: T
authors.lastname: Funaki
authors.forename: Akira
authors.initials: A
authors.lastname: Matsuda
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Watanabe
authors.forename: Liang
authors.initials: L
authors.lastname: Ning
authors.forename: Zhuo
authors.initials: Z
authors.lastname: Xu
authors.forename: Akira
authors.initials: A
authors.lastname: Murakami
authors.forename: Eri
authors.initials: E
authors.lastname: Arikawa-Hirasawa
mesh_terms: D000818:Animals; D004247:DNA / Q000235:genetics*; D004195:Disease Models, Animal; D019573:Epithelium, Corneal / Q000378:metabolism / Q000648:ultrastructure*; D019812:Heparan Sulfate Proteoglycans / Q000235:genetics* / Q000378:metabolism; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D051379:Mice; D008822:Mice, Transgenic; D008850:Microphthalmos / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D046529:Microscopy, Electron, Transmission; D009154:Mutation; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D019812:Heparan Sulfate Proteoglycans; C071927:perlecan; D004247:DNA