Inhibition of Ephrin-B2 in brain pericytes decreases cerebral pathological neovascularization in diabetic rats

Abstract

We have previously shown that diabetes causes dysfunctional cerebral neovascularization that increases the risk for cerebrovascular disorders such as stroke and cognitive impairment. Pericytes (PCs) play a pivotal role in the angiogenic process through their interaction with the endothelial cells (EC). Yet, the role of PCs in dysfunctional cerebral neovascularization in diabetes is unclear. In the present study, we tested the hypothesis that the increased proangiogenic Ephrin-B2 signaling in PCs contributes to the dysfunctional cerebral neovascularization in diabetes. Type-II diabetes was induced by a combination of high fat diet and low dose streptozotocin injection in male Wistar rats. Selective in vivo Ephrin-B2 silencing in brain PCs was achieved using the stereotactic injection of adeno-associated virus (AAV) with NG2-promoter that expresses Ephrin-B2 shRNA. Neovascularization was assessed using vascular fluorescent dye stain. Novel object recognition (NOR) test was used to determine cognitive functions. Human brain microvascular pericytes HBMVPCs were grown in high glucose 25 mM and palmitate 200 uM (HG/Pal) to mimic diabetic conditions. Scratch migration and tube formation assays were conducted to evaluate PC/EC interaction and angiogenic functions in PC/EC co-culture. Diabetes increased the expression of Ephrin-B2 in the cerebrovasculature and pericytes. Concomitant increases in cerebral neovascularization parameters including vascular density, tortuosity and branching density in diabetic rats were accompanied by deterioration of cognitive function. Inhibition of Ephrin-B2 expression in PCs significantly restored cerebral vascularization and improved cognitive functions. HG/Pal increased PC/EC angiogenic properties in co-culture. Silencing Ephrin-B2 in PCs significantly reduced PC migration and PC/EC co-culture angiogenic properties. This study emphasizes the significant contribution of PCs to the pathological neovascularization in diabetes. Our findings introduce Ephrin-B2 signaling as a promising therapeutic target to improve cerebrovascular integrity in diabetes.

Fig 1. Increased Ephrin-B2 expression in pericytes in diabetes.

Four weeks male Wistar rats were injected with low dose streptozotocin followed by 8-weeks of high-fat diet (HFD, 45% fat). Brains were isolated and fixed. Brain sections were reacted with anti-Ephrin-B2 antibody and Iso-Lectin-B4. (A) Co-localized Ephrin-B2 (Blue), PDGFR-β (green) on Iso-Lectin-B4 (red) was compared between diabetic and control Wistar rats. Diabetic rats showed increased Ephrin-B2 expression in perivascular area that was co-localized with the pericytes marker. (B) Quantification of the relative optical density. (C) Western blot analysis showed a significant increase in Ephrin-B2 expression in the brain cortex of diabetic rats compared to control. (N = 4–5, *P<0.05 vs control).

Fig 2. Diabetic conditions increases Ephrin-B2 signaling in pericytes.

Human brain microvascular pericyte cells were treated with high glucose (25 mM) and sodium palmitate (200 uM) to mimic diabetic conditions. Equimolar of L-glucose was used as an osmotic control. A) Western blot analysis showed a significant increase in Ephrin-B2 expression and activation in pericytes under diabetic conditions. Moreover, results showed that diabetes caused an increase in downstream angiogenic signal as illustrated with enhanced focal adhesion kinase activation in pericytes. (*P<0.05 vs NG, n = 4). B) Pericytes were treated with high glucose (25mM glucose) and sodium palmitate (200 uM) showed 2 fold increase in cell migration assay. Silencing Ephrin-B2 was achieved using Ephrin-B2 siRNA. Silencing Ephrin-B2 in pericytes abolished diabetes-induced increase migration. (Two way ANOVA, Gene vs diabetic condition, significance: yes, Interaction: yes, n = 3 duplicate).

Fig 3. Silencing Ephrin-B2 expression in pericytes decreased diabetes-mediated angiogenic signaling.

Ephrin-B2 was silenced in human brain microvascular pericytes using siRNA technology. Human brain microvascular pericytes and endothelial cells were grown in ratio one to four respectively in normal glucose or high glucose (25 mM) and palmitate (200 uM) to mimic diabetic conditions. L-glucose was used as an osmotic control. Representative images and quantifications from matrigel tube formation and filopodia extensions in pericytes/endothelial co-cultures are shown in panels A and B. Our results shows a significant increase in tube formation and number of filopodia extensions in diabetic conditions compared to control. Silencing Ephrin-B2 expression in pericytes abolished diabetes induced angiogenic signaling in pericytes/endothelial cell co-culture. (Two way ANOVA, Gene vs diabetic condition, significance: yes, Interaction: yes, n = 3 duplicate).

Fig 4. Selective reduction of Ephrin-B2 expression in pericytes in vivo.

A) Ephrin-B2 expression was significantly reduced in human brain microvascular pericytes using adeno-associated virus (AAV) the express Ephrin-B2 shRNA under NG-2 promotor, a pericyte promotor. Immunoblotting studies confirmed significant reduction of Ephrin-B2 in pericytes. Pericytes expressed green fluoresce protein as another indication of successful transfection. (*P<0.05 vs Control, n = 3). (B) Immunoblotting studies showed a decrease in Ephrin-B2 expression in ipsilateral hemisphere after stereotactic injection of the AAV into striatum compared to the contralateral side. (*P<0.05 vs contralateral, n = 3). (C) Representatives of immunohistochemical studies showing the localization of the AAV green fluoresce protein expression with only the pericyte marker, PDGFR-β, but not the endothelial cell marker, iso-lectin B4 following intraocular injection of the AAV virus.

Fig 5. Silencing Ephrin-B2 in pericytes in diabetic rats restored cerebral vascularization and improved cognitive functions.

Ephrin-B2 was decreased in cerebral pericytes using AAV viral injection. Animals were perfused with vascular Dil stain. (A) Representatives of brain sections imaged using confocal microscopy. 3D images reconstructs were quantified for neovascularization indices (B) vascular density (C) tortuosity and (D) branch density. Diabetes-induced pathological neovascularization, and selective inhibition of Ephrin-B2 in pericytes restored the neovascularization indices in diabetic rats. E) Novel object recognition test showing cognitive deterioration of diabetic animals compared to control. Selective inhibition of Ephrin-B2 in pericytes improved cognitive functions in diabetes. (N = 4–6, *P<0.05 vs control, ^#P<0.05 vs diabetes).

Fig 6. Increased Ephrin-B2 signaling contributes to diabetes-induced cerebral pathological neovascularization.

Increased Ephrin-B2 signaling in pericytes in diabetes enhances pericytes migration and impairs pericytes/endothelial cells interactions leading to a decrease in vascular stability and pathological cerebral neovascularization.