Enhanced VEGF signalling mediates cerebral neovascularisation via downregulation of guidance protein ROBO4 in a rat model of diabetes

Aims/hypothesis 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. Methods 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. Results ROBO4 expression was significantly decreased in the cerebral vasculature as well as in BMVECs in diabetes ( p