title: Angiopoietin-1 is regulated by miR-204 and contributes to corneal neovascularization in KLEIP-deficient mice.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Vascular Biology and Tumor Angiogenesis, Center for Biomedicine and Medical Technology Mannheim (CBTM), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Division of Vascular Oncology and Metastasis, German Cancer Research Center (DKFZ-ZMBH Alliance), Heidelberg, Germany.
authors.forename: Jakob N
authors.initials: JN
authors.lastname: Kather
authors.affiliation: 5th Medical Department, University Hospital Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany.
authors.forename: Julian
authors.initials: J
authors.lastname: Friedrich
authors.affiliation: Department of Vascular Biology and Tumor Angiogenesis, Center for Biomedicine and Medical Technology Mannheim (CBTM), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Division of Vascular Oncology and Metastasis, German Cancer Research Center (DKFZ-ZMBH Alliance), Heidelberg, Germany.
authors.forename: Nicole
authors.initials: N
authors.lastname: Woik
authors.affiliation: Medical Research Center, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Sticht
authors.affiliation: Medical Research Center, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany.
authors.forename: Norbert
authors.initials: N
authors.lastname: Gretz
authors.affiliation: 5th Medical Department, University Hospital Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany.
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Hammes
authors.affiliation: Department of Vascular Biology and Tumor Angiogenesis, Center for Biomedicine and Medical Technology Mannheim (CBTM), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Division of Vascular Oncology and Metastasis, German Cancer Research Center (DKFZ-ZMBH Alliance), Heidelberg, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Kroll
mesh_terms: D020533:Angiogenesis Inhibitors / Q000008:administration & dosage; D042683:Angiopoietin-1 / Q000096:biosynthesis / Q000235:genetics*; D000818:Animals; D015153:Blotting, Western; D016510:Corneal Neovascularization / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000172:deficiency*; D004195:Disease Models, Animal; D005786:Gene Expression Regulation; D007150:Immunohistochemistry; D058449:Intravitreal Injections; D051379:Mice; D018345:Mice, Knockout; D035683:MicroRNAs / Q000096:biosynthesis / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis; D010641:Phenotype; D012333:RNA, Messenger / Q000235:genetics*; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors
chemical_list: D020533:Angiogenesis Inhibitors; D042683:Angiopoietin-1; D004268:DNA-Binding Proteins; C574692:MIRN204 microRNA, mouse; D035683:MicroRNAs; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; C522548:kleisen beta protein, mouse
keywords: KLEIP; KLHL20; angiopoietin-1; corneal neovascularization; miR-204