title: Blockade of insulin receptor substrate-1 inhibits corneal lymphangiogenesis.
abstract: OBJECTIVE
  To analyze whether insulin receptor substrate (IRS-1) is involved in lymphatic vessel development and whether IRS-1 blockade can inhibit lymphangiogenesis in vivo.
  
  
  METHODS
  The impact of IRS-1 blockade by GS-101 (Aganirsen), an antisense oligonucleotide against IRS-1, on lymphatic endothelial cell (LEC) proliferation was assessed by ELISA. Furthermore, the effect of IRS-1 blockade on prolymphangiogenic growth factor expression by LECs and macrophages (peritoneal exudate cells) was tested by real-time PCR. The mouse model of inflammatory corneal neovascularization was used to analyze the effect of IRS-1 blockade in vivo: after corneal suture placement, mice were treated with GS-101 eye drops (twice daily afterwards for 1 week, 5 μL per drop; 50, 100, or 200 μM). Afterward, corneal wholemounts were prepared and stained for blood and lymphatic vessels.
  
  
  RESULTS
  Blockade of IRS-1 by GS-101 inhibited LEC proliferation dose dependently. GS-101 led to decreased VEGF-A expression levels in LECs, whereas VEGF-C, VEGF-D, and VEGFR3 showed no significant change. In macrophages, VEGF-A expression levels were also inhibited by IRS-1 blockade. Additionally, GS-101 strongly inhibited macrophage-derived VEGF-C, VEGF-D, and VEGFR3 expression. In vivo, corneal hemangiogenesis was significantly inhibited when used at a concentration of 200 μM (by 17%; P < 0.01). Corneal lymphangiogenesis was significantly inhibited when used at a dose of 100 μM (by 21%; P < 0.01), and the highest used dose (200 μM) showed an even stronger inhibition (by 28%; P < 0.001).
  
  
  CONCLUSIONS
  Blockade of IRS-1 inhibits not only hemangiogenesis but also lymphangiogenesis. To the authors' knowledge, this is the first evidence that IRS-1 is involved in the molecular pathway leading to lymphangiogenesis.
authors.affiliation: Department of Ophthalmology, University of Cologne, Cologne, Germany.
authors.forename: Deniz
authors.initials: D
authors.lastname: Hos
authors.forename: Birgit
authors.initials: B
authors.lastname: Regenfuss
authors.forename: Felix
authors.initials: F
authors.lastname: Bock
authors.forename: Jasmine
authors.initials: J
authors.lastname: Onderka
authors.forename: Claus
authors.initials: C
authors.lastname: Cursiefen
mesh_terms: D000818:Animals; D002455:Cell Division / Q000502:physiology; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000502:physiology*; D004728:Endothelium, Corneal / Q000166:cytology / Q000502:physiology*; D005260:Female; D006801:Humans; D055504:Insulin Receptor Substrate Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D042583:Lymphangiogenesis / Q000502:physiology*; D017737:Macrophages, Peritoneal / Q000166:cytology / Q000502:physiology*; D051379:Mice; D009841:Oligonucleotides / Q000494:pharmacology; D009883:Ophthalmic Solutions; D015398:Signal Transduction / Q000502:physiology; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics; D042582:Vascular Endothelial Growth Factor C / Q000235:genetics; D042643:Vascular Endothelial Growth Factor D / Q000235:genetics; D040321:Vascular Endothelial Growth Factor Receptor-3 / Q000235:genetics
chemical_list: C540750:GS 101; C526292:IRS1 protein, human; D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; D009841:Oligonucleotides; D009883:Ophthalmic Solutions; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D042582:Vascular Endothelial Growth Factor C; D042643:Vascular Endothelial Growth Factor D; C467480:vascular endothelial growth factor A, mouse; D040321:Vascular Endothelial Growth Factor Receptor-3