title: Ex vivo transfer of Smad7 decreases damage to the corneal endothelium after penetrating keratoplasty.
abstract: OBJECTIVE
  To determine whether transient gene transfer and expression of the intracellular antagonist of transforming growth factor beta (TGF-beta), Smad7, to corneal endothelial cells decreases corneal endothelial cell damage after penetrating keratoplasty in a rabbit model.
  
  
  METHODS
  Rabbit corneas were transfected ex vivo with replication-deficient adenoviruses encoding Flag-tagged Smad7, Flag-tagged Smad3, or LacZ (termed AdCMV-Smad7, AdCMV-Smad3, AdCMV-LacZ) and then transplanted to normal rabbits. Expression of the exogenous Smads and phosphorylation of endogenous Smad2 in the transplanted corneal endothelium were examined by immunoblotting and immunohistochemistry with anti-Flag or anti-phosphorylated Smad2 antibodies. Cellular density and morphological changes in the corneal endothelium of the transplanted cornea were evaluated by scanning electron microscopy after transplantation of the Smad-transfected corneas.
  
  
  RESULTS
  Transplanted AdCMV-Smad7-transfected corneas significantly inhibited the decrease in cellular density and accelerated wound healing at the host-graft junction when compared with transplanted AdCMV-LacZ-transfected corneas. Transplanted AdCMV-Smad3-transfected corneas showed decreased cellular density and delayed wound healing at the host-graft junction.
  
  
  CONCLUSIONS
  Ex vivo gene transfer of Smad7 to corneal endothelial cells inhibits the decrease in cellular density and accelerates wound healing after penetrating keratoplasty in rabbits. Thus, modulation of Smad7 expression in corneal endothelial cells may decrease corneal endothelial cell damage after penetrating keratoplasty.
authors.affiliation: Department of Ophthalmology, Juntendo University School of Medicine, Tokyo, Japan.
authors.forename: Toshinari
authors.initials: T
authors.lastname: Funaki
authors.affiliation: Department of Ophthalmology, Juntendo University School of Medicine, Tokyo, Japan. ebihara@med.juntendo.ac.jp.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Ebihara
authors.affiliation: Department of Ophthalmology, Juntendo University School of Medicine, Tokyo, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Murakami
authors.affiliation: Department of Immunology, University of Yamanashi Faculty of Medicine, Yamanashi, Japan.
authors.forename: Atsuhito
authors.initials: A
authors.lastname: Nakao
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001082:Aqueous Humor / Q000378:metabolism; D015153:Blotting, Western; D002452:Cell Count; D002470:Cell Survival / Q000502:physiology; D004195:Disease Models, Animal; D004728:Endothelium, Corneal / Q000378:metabolism* / Q000648:ultrastructure; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D005786:Gene Expression Regulation / Q000502:physiology*; D005822:Genetic Vectors; D015948:Keratoplasty, Penetrating; D008855:Microscopy, Electron, Scanning; D010766:Phosphorylation; D011817:Rabbits; D051899:Smad2 Protein / Q000378:metabolism; D051900:Smad3 Protein / Q000235:genetics; D051906:Smad7 Protein / Q000235:genetics*; D014162:Transfection; D053781:Transforming Growth Factor beta2 / Q000378:metabolism; D014945:Wound Healing / Q000502:physiology*
chemical_list: D051899:Smad2 Protein; D051900:Smad3 Protein; D051906:Smad7 Protein; D053781:Transforming Growth Factor beta2