title: Angiopoietin-like 7 is an anti-angiogenic protein required to prevent vascularization of the cornea.
abstract: OBJECTIVE
  We sought to identify the anti-angiogenic molecule expressed in corneal keratocytes that is responsible for maintaining the avascularity of the cornea.
  
  
  METHODS
  Human umbilical vein endothelial cells (HUVECs) were cultured with either human dermal fibroblasts or with human corneal keratocytes under serum-free conditions. The areas that exhibited blood vessel formation were estimated by immunostaining the cultures with an antitibody against CD31, a blood vessel marker. We also performed microarray gene-expression analysis and selected one molecule, angiopoietin-like 7 (ANGPTL7) for further functional studies conducted with the keratocytes and in vivo in mice.
  
  
  RESULTS
  Areas showing blood vessel formation in normal serum-free medium were conditions were markedly smaller when HUVECs were co-cultured with corneal keratocytes than when they were co-cultured with the dermal fibroblasts under the same conditions. Microarray analysis revealed that ANGPTL7 expression was higher in keratocytes than in dermal fibroblasts. In vitro, inhibiting ANGPTL7 expression by using a specific siRNA led to greater tube formation than did the transfection of cells with a control siRNA, and this increase in tube formation was abolished when recombinant ANGPTL7 protein was added to the cultures. In vivo, intrastromal injections of an ANGPTL7 PshRNA into the avascular corneal stroma of mice resulted in the growth of blood vessels.
  
  
  CONCLUSIONS
  ANGPTL7, which is abundantly expressed in keratocytes, plays a major role in maintaining corneal avascularity and transparency.
authors.affiliation: Department of Ophthalmology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Toyono
authors.affiliation: Department of Ophthalmology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.
authors.forename: Tomohiko
authors.initials: T
authors.lastname: Usui
authors.affiliation: Department of Ophthalmology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.
authors.forename: Seiichi
authors.initials: S
authors.lastname: Yokoo
authors.affiliation: Department of Ophthalmology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.
authors.forename: Yukako
authors.initials: Y
authors.lastname: Taketani
authors.affiliation: Department of Ophthalmology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.
authors.forename: Suguru
authors.initials: S
authors.lastname: Nakagawa
authors.affiliation: Department of Pathology, Tokyo Medical University, Tokyo, Japan.
authors.forename: Masahiko
authors.initials: M
authors.lastname: Kuroda
authors.affiliation: Department of Ophthalmology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.
authors.forename: Satoru
authors.initials: S
authors.lastname: Yamagami
authors.affiliation: Department of Ophthalmology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.
authors.forename: Shiro
authors.initials: S
authors.lastname: Amano
mesh_terms: D000091307:Angiopoietin-Like Protein 7; D000075802:Angiopoietin-like Proteins; D042682:Angiopoietins / Q000502:physiology*; D000818:Animals; D002478:Cells, Cultured; D018920:Coculture Techniques; D016510:Corneal Neovascularization / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism; D008297:Male; D008810:Mice, Inbred C57BL; D059467:Transcriptome
chemical_list: C530622:ANGPTL7 protein, human; D000091307:Angiopoietin-Like Protein 7; D000075802:Angiopoietin-like Proteins; D042682:Angiopoietins