title: Enhanced VEGF signalling mediates cerebral neovascularisation via downregulation of guidance protein ROBO4 in a rat model of diabetes.
abstract: Diabetes promotes cerebral neovascularisation via increased vascular endothelial growth factor (VEGF) angiogenic signalling. Roundabout-4 (ROBO4) protein is an endogenous inhibitor of VEGF signalling that stabilises the vasculature. Yet, how diabetes affects ROBO4 function remains unknown. We hypothesised that increased VEGF signalling in diabetes decreases ROBO4 expression and function via binding of ROBO4 with VEGF-activated β3 integrin and that restoration of ROBO4 expression prevents/repairs cerebral neovascularisation in diabetes.
  
  
  
  ROBO4 protein expression in a rat model of type 2 diabetes (Goto-Kakizaki [GK] rats) was examined by western blotting and immunohistochemistry. ROBO4 was locally overexpressed in the brain and in primary brain microvascular endothelial cells (BMVECs). GK rats were treated with SKLB1002, a selective VEGF receptor-2 (VEGFR-2) antagonist. Cerebrovascular neovascularisation indices were determined using a FITC vascular space-filling model. Immunoprecipitation was used to determine ROBO4-β3 integrin interaction.
  
  
  
  ROBO4 expression was significantly decreased in the cerebral vasculature as well as in BMVECs in diabetes (p < 0.05). Silencing Robo4 increased the angiogenic properties of control BMVECs (p < 0.05). In vivo and in vitro overexpression of ROBO4 inhibited VEGF-induced angiogenic signalling and increased vessel maturation. Inhibition of VEGF signalling using SKLB1002 increased ROBO4 expression (p < 0.05) and reduced neovascularisation indices (p < 0.05). Furthermore, SKLB1002 significantly decreased ROBO4-β3 integrin interaction in diabetes (p < 0.05).
  
  
  
  Our study identifies the restoration of ROBO4 and inhibition of VEGF signalling as treatment strategies for diabetes-induced cerebral neovascularisation.
authors.affiliation: Charlie Norwood Veterans Administration Medical Center, Augusta, GA, USA. mabdelsaid@augusta.edu.|Department of Physiology, Augusta University, 1120 15th Street CA-3135, Augusta, GA, 30912, USA. mabdelsaid@augusta.edu.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Abdelsaid
authors.affiliation: Charlie Norwood Veterans Administration Medical Center, Augusta, GA, USA.|Department of Physiology, Augusta University, 1120 15th Street CA-3135, Augusta, GA, 30912, USA.
authors.forename: Maha
authors.initials: M
authors.lastname: Coucha
authors.affiliation: Charlie Norwood Veterans Administration Medical Center, Augusta, GA, USA.|Department of Physiology, Augusta University, 1120 15th Street CA-3135, Augusta, GA, 30912, USA.
authors.forename: Sherif
authors.initials: S
authors.lastname: Hafez
authors.affiliation: Charlie Norwood Veterans Administration Medical Center, Augusta, GA, USA.|Department of Physiology, Augusta University, 1120 15th Street CA-3135, Augusta, GA, 30912, USA.
authors.forename: Abdul
authors.initials: A
authors.lastname: Yasir
authors.affiliation: Department of Biostatistics, Augusta University, Augusta, GA, USA.
authors.forename: Maribeth H
authors.initials: MH
authors.lastname: Johnson
authors.affiliation: Charlie Norwood Veterans Administration Medical Center, Augusta, GA, USA.|Department of Physiology, Augusta University, 1120 15th Street CA-3135, Augusta, GA, 30912, USA.
authors.forename: Adviye
authors.initials: A
authors.lastname: Ergul
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism; D003924:Diabetes Mellitus, Type 2 / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation / Q000235:genetics / Q000502:physiology; D009389:Neovascularization, Pathologic / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*; D000097905:Roundabout Proteins
chemical_list: D011956:Receptors, Cell Surface; D042461:Vascular Endothelial Growth Factor A; C578845:Robo4 protein, rat; D000097905:Roundabout Proteins
keywords: Angiogenesis; Anti-VEGF; Brain neovascularisation; Diabetes; ROBO4 signalling; VEGF signalling