Diabetes increases cerebrovascular complications such as stroke and cognitive impairment. We have previously showed that diabetes causes dysfunctional cerebral neovascularization. Pericytes have a pivotal role in modulating microvascular physiology and pathology, yet its role in diabetes dysfunctional cerebral neovascularization is unknown. In the present study we testedthe hypothesis that pericytes play a crucial role in diabetes-mediated dysfunctional pathological cerebral neovascularization via dysregulation of antiangiogenic Slit-2/ROBO4 and the proangiogenic EphrinB2/EphB4 signaling.
Methods: Diabetes was induced in Wistar rats using low dose of streptozotocin and high fat diet (STZ/HFD). Immunohistochemistry and immunoblotting were used to assess ROBO4 and EphrinB2 expression in the brain and human brain microvascular pericytes (HBMVP). HBMVP was exposed to glucose (25 mM) /palmitate (200 uM) to mimic diabetic conditions. Scratch migration assay was used to assess the effect of high glucose/palmitate (HG/Pal) on pericytes migration. Adenovirus was used to overexpress ROBO4 in the brain in vivo.
Results: STZ/HFD caused a significant increase in HbA1C (10.8*). Diabetic brains and HBMVP exposed to HG/Pal showed reduced ROBO4 expression (66%* and 48%*) and increased EphrinB2 expression (1.9* and 1.6* folds). HG/Pal increased pericyte migration by 2* fold. Pericyte migration was increased by 2.5* fold with silencing ROBO4 but significantly reduced with silencing EphrinB2 (35%*) or ROBO4 overexpression (47%*) under HG/Pal. In vivo ROBO4 overexpression decreased pathological neovascularization indices and increased pericyte coverage. (n= 3-5, *P<0.05).
Conclusion: Our findings emphasize the importance of balanced anti- and proangiogenic signaling for proper pericyte function in diabetes. Our results suggest that pericytes are involved in proangiogenic and barrier functions of endothelial cells and pericyte dysfunction may contribute to pathological neovascularization in diabetes. Thus, pericyte ROBO4 and EphrinB2 signaling are promising therapeutic targets to improve cerebrovascular integrity in diabetes.