Abstract
Diabetes increases the risk and exacerbates the progress of cerebral complications including stroke and cognitive impairment. Diabetic Goto-Kakizaki (GK) rats suffer loss of vascular integrity and dysfunctional cerebral-neovascularization, despite augmented vascular endothelial growth factor (VEGF). Roundabout-4 (Robo-4) is an endothelial cell (EC) specific member axon guidance molecule that regulates EC stability via inhibiting VEGF signaling but its role in cerebrovascular maturation in diabetes remained unknown. We hypothesized that increased VEGF activation in diabetes reduces Robo-4 and uncouples its protective signaling by promoting its binding to activated β-3 integrin. Methods: Immunohistochemistry and immunoblotting are used to assess Robo-4 expression, pericyte/EC ratio and robo-4/β-3 integrin interaction in the brain and isolated microvascular endothelial cells (BMVEC). Transfection was used to overexpress Robo-4 in BMVEC in vitro and EC migration and trans-well permeability assays were used to examine EC function. Stereotaxic injection of adenoviral construct was used for local in vivo overexpression of robo-4 and its impact on neovascularization indices (Vascular Volume (VV), Surface area (SA), Tortuosity index (TI) and Vascular density (VD) was measured using 3D confocal microscopy. Results: Robo-4 expression was reduced in GK brains as well as in isolated BMVEC (35%* and 39%* vs control Wistar). In vitro, over expression of Robo-4 inhibited VEGF-induced permeability (80%*) and cell migration (70%*) in BMVEC vs control. In vivo over expression of Robo-4 improved vascular integrity and maturation as indicated by reduction of all pathological neovascularization indices (VV:70%*, SA:50%*, TI:20%*, VD:50%* vs control) and increased pericyte/EC ratio (40%* vs control). GK rats showed increase interaction between VEGF-activated β-3 integrin and Robo-4. Overexpression of Robo-4 decreased this interaction (40%* vs control). (*P<0.05). Conclusion: Restoration of Robo-4 expression decreased pathological neovascularization and improved vessel integrity and maturation. Our results suggest that Robo-4 is a promising therapeutic target in treatment of diabetic pathological brain neovascularization.
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