title: Keratocytes create stromal spaces to promote corneal neovascularization via MMP13 expression.
abstract: OBJECTIVE
  To investigate the exact mechanism by which keratocytes promote corneal neovascularization.
  
  
  METHODS
  The expression of matrix metalloproteinase 13 (MMP13), cluster of differentiation 146 (CD146), VEGFa, VEGFc, VEGF receptor (r)2, and VEGFr3 by normal and alkali-burned rat corneas was determined via quantitative (q)RT-PCR and/or Western blot analysis or in situ hybridization. Corneal neovascularization was observed under a slit lamp microscope and evaluated via immunohistochemistry. The cells that expressed MMP13 in the corneas were determined via sequential immunohistochemistry and in situ hybridization. The degradation of type I collagen was evaluated via the detection of hydroxyproline content and Western blot analysis. The effects of VEGFa and VEGFc on MMP13 expression were determined via luciferase reporter assay for the MMP13 promoter and primary keratocyte culture.
  
  
  RESULTS
  Matrix metalloproteinase 13 was predominantly expressed by epithelial cells in normal rat corneas, but it was expressed by cells in corneal stromas after alkali burns. The formation of new blood vessels was consistent with MMP13 expression and attenuated by a selective MMP13 inhibitor in alkali-burned corneas. Keratocytes were the major cells expressing MMP13 in corneal stromas after alkali burns. Through MMP13 expression, keratocytes directly degraded collagen type I to create stromal spaces, which were convenient for newly formed blood vessels to grow into. Expression of MMP13 and collagen type I degradation via keratocytes were induced by VEGFc through VEGFr3 and inhibited by antibodies for VEGFc and VEGFr3.
  
  
  CONCLUSIONS
  Keratocytes could directly degrade type I collagen and create stromal spaces, promoting corneal neovascularization through VEGFc/VEGFr3-induced MMP13 expression.
authors.affiliation: Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Ma
authors.affiliation: Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Zhou
authors.affiliation: Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China.
authors.forename: Meihua
authors.initials: M
authors.lastname: Fan
authors.affiliation: Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China.
authors.forename: He
authors.initials: H
authors.lastname: Wang
authors.affiliation: Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China.
authors.forename: Caihong
authors.initials: C
authors.lastname: Huang
authors.affiliation: Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China.
authors.forename: Zhaoqiang
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China Affiliated Xiamen Eye Center of Xiamen University, Xiamen, Fujian, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.affiliation: Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China.
authors.forename: Yongxiong
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China Affiliated Xiamen Eye Center of Xiamen University, Xiamen, Fujian, China.
authors.forename: Zuguo
authors.initials: Z
authors.lastname: Liu
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D060527:Corneal Keratocytes / Q000201:enzymology / Q000473:pathology*; D016510:Corneal Neovascularization / Q000201:enzymology / Q000235:genetics* / Q000473:pathology; D004247:DNA / Q000235:genetics*; D004195:Disease Models, Animal; D005786:Gene Expression Regulation; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D008297:Male; D053509:Matrix Metalloproteinase 13 / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice; D051381:Rats; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D004247:DNA; D053509:Matrix Metalloproteinase 13; C507083:Mmp13 protein, rat
keywords: VEGFc; VEGFr3; corneal neovascularization; keratocytes; metalloproteinases-13; type I collagen