Angiopoietin-1 is regulated by miR-204 and contributes to corneal neovascularization in KLEIP-deficient mice

Invest Ophthalmol Vis Sci. 2014 Jun 10;55(7):4295-303. doi: 10.1167/iovs.13-13619.

Abstract

Purpose: Corneal neovascularization can cause loss of vision. The introduction of anti-VEGF therapy has been a major improvement in therapeutic options. Recently, we established Kelch-like Ect2-interacting protein (KLEIP/KLHL20) knockout mice as a model of spontaneous corneal neovascular dystrophy. The aim of the present study was to characterize corneal neovascularization in progressive corneal dystrophy in KLEIP(-/-) mice, to evaluate the efficacy of anti-VEGF therapy, and to identify novel molecular regulators in this experimental model.

Methods: Corneal neovascularization was assessed by immunohistochemistry. Vascular endothelial growth factor signaling was inhibited by injection of a blocking antibody. Microarrays were used to measure expression of mRNA and microRNA (miRNA) in dystrophic corneae. Results were validated by immunohistochemistry and Western blotting.

Results: Blood vessels and lymphatics grew from the limbus toward the dystrophic epithelium in corneae of KLEIP(-/-) mice. Blocking VEGF signaling did not reduce phenotype progression. Correspondingly, microarray analysis revealed no upregulation of canonical vascular growth factors in late dystrophy. During phenotype progression, angiopoietin-1 expression increased while miR-204 expression decreased. Bioinformatic analysis identified a binding site for miR-204 in the angiopoietin-1 gene. Validation by in vitro experiments confirmed regulation of angiopoietin-1 by miR-204.

Conclusions: Vascular endothelial growth factor does not act as a major player in corneal neovascularization in KLEIP(-/-) mice. However, the proangiogenic factor angiopoietin-1 was strongly upregulated in late-stage phenotype, correlating with loss of miR-204 expression. Correspondingly, we identified miR-204 as a novel regulator of angiopoietin-1 in vitro. These findings may explain the incomplete efficacy of anti-VEGF therapy in the clinic and may provide new candidates for pharmaceutical intervention.

Keywords: KLEIP; KLHL20; angiopoietin-1; corneal neovascularization; miR-204.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Angiogenesis Inhibitors / administration & dosage
  • Angiopoietin-1 / biosynthesis
  • Angiopoietin-1 / genetics*
  • Animals
  • Blotting, Western
  • Corneal Neovascularization / drug therapy
  • Corneal Neovascularization / genetics*
  • Corneal Neovascularization / metabolism
  • DNA-Binding Proteins / deficiency*
  • Disease Models, Animal
  • Gene Expression Regulation*
  • Immunohistochemistry
  • Intravitreal Injections
  • Mice
  • Mice, Knockout
  • MicroRNAs / biosynthesis
  • MicroRNAs / genetics*
  • Oligonucleotide Array Sequence Analysis
  • Phenotype
  • RNA, Messenger / genetics*
  • Vascular Endothelial Growth Factor A / antagonists & inhibitors

Substances

  • Angiogenesis Inhibitors
  • Angiopoietin-1
  • DNA-Binding Proteins
  • MIRN204 microRNA, mouse
  • MicroRNAs
  • RNA, Messenger
  • Vascular Endothelial Growth Factor A
  • kleisen beta protein, mouse