robo4 adenovirus  (Vector Biolabs)

Journal: Diabetologia

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

doi: 10.1007/s00125-017-4214-6

Figure Lengend Snippet: ROBO4 regulates VEGF-induced angiogenic signal. (a) Silencing Robo4 in BMVECs isolated from control Wistar rats (Wis, light grey bars) resulted in a significant increase in migration and tube formation compared with cells transfected with scrambled (Sc) siRNA (white bars). BMVECs isolated from diabetic GK rats (black bars) showed significant increases in migration and tube formation. Overexpression of ROBO4 in diabetic BMVECs (dark grey bars) significantly decreased endothelial cell migration and tube formation (n=3 in duplicate, *p<0.05 vs Wistar+Sc siRNA, †p<0.05 vs GK+GFP). (b) Transwell permeability assay showed that overexpression of ROBO4 (black bars) significantly decreased BMVEC permeability in diabetes compared with control (n=3 in duplicate, †p<0.05 vs GK+GFP). (c) Representative image of brain and adenovirus stereotactic injection site. ROBO4 was overexpressed by 50% compared with empty vector control. (n=3, *p<0.05 vs empty vector). Scale bar, 10 μm. (d) Representative image and quantification of neovascularisation indices after ROBO4 overexpression. ROBO4 overexpression (Robo4 AD) significantly reduced all neovascularisation indices (n=4, *p<0.05 vs contralateral side). Scale bars, 50 μm

Article Snippet: ROBO4 overexpression In vivo experiments Robo4 adenovirus (3 μl of 1.85 × 10 12 viral particles/ml, Ad- Gfp -h Robo4 , ADV-221472; Vector Biolabs, Philadelphia, PA, USA) was injected over 6 min via a 30-gauge needle adjacent to the MCA at stereotactic coordinates (bregma: anterior–posterior, −0.5 mm; mediolateral −1 mm; dorsoventral, −4 mm).

Techniques: Isolation, Control, Migration, Transfection, Over Expression, Permeability, Injection, Plasmid Preparation

Journal: Diabetologia

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

doi: 10.1007/s00125-017-4214-6

Figure Lengend Snippet: VEGFR-2 inhibitor, SKLB1002, increases ROBO4 availability. Diabetic GK rats were treated with vehicle or SLKB1002 (5, 10 or 15 mg/kg per day i.p.) for 2 weeks. (a) Western blot analysis of VEGFR-2 activation. GK rats treated with SLKB1002 showed significant decrease in VEGFR-2 phosphorylation compared with control vehicle-treated rats (n=3, *p<0.05 vs vehicle). (b) Western blot analysis of ROBO4 expression in GK rats treated with SLKB1002 showed increased expression of ROBO4 (n=3 or 4, *p<0.05 vs vehicle). (c) Western blot analysis of VEGF-induced phosphorylation of β3 integrin (β-3). Treatment with SLKB1002 or ROBO4 overexpression (ROBO4 AD) significantly reduced VEGF-induced phosphorylation of β3 integrin in GK rats. (n=3 or 4, *p<0.05 vs GK). (d) Immunoprecipitation (IP) of brain homogenate from GK rats treated with SKLB1002 or overexpressing ROBO4. Both treatments significantly decreased the ROBO4–β3 integrin interaction and binding in GK brain homogenate (n=3 or 4, *p<0.05 vs GK). (e) Representative images and quantification of cell migration and tube formation. Dashed white lines represent migration after 18 h. Silencing the β3 integrin gene significantly decreased cellular tube formation and migration in BMVECs isolated from diabetic GK rats compared with GK BMVECs treated with scrambled siRNA (Sc siRNA) (n=3 in duplicate, *p<0.05 vs GK+Sc siRNA)

Article Snippet: ROBO4 overexpression In vivo experiments Robo4 adenovirus (3 μl of 1.85 × 10 12 viral particles/ml, Ad- Gfp -h Robo4 , ADV-221472; Vector Biolabs, Philadelphia, PA, USA) was injected over 6 min via a 30-gauge needle adjacent to the MCA at stereotactic coordinates (bregma: anterior–posterior, −0.5 mm; mediolateral −1 mm; dorsoventral, −4 mm).

Techniques: Western Blot, Activation Assay, Phospho-proteomics, Control, Expressing, Over Expression, Immunoprecipitation, Binding Assay, Migration, Isolation

Journal: Diabetologia

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

doi: 10.1007/s00125-017-4214-6

Figure Lengend Snippet: Diabetes decreases the expression of ROBO4 in cerebral vasculature. Diabetic GK rats were injected with FITC–dextran via the jugular vein. Brain sections were reacted with anti-ROBO4 antibody. (a) Co-localised ROBO4 (red) on FITC-filled cerebral vasculature (green) was compared between diabetic GK rats and control Wistar rats. Diabetic GK rats showed decreased ROBO4 expression (n=4 or 5, *p<0.05 vs Wistar). (b) Western blot of BMVECs isolated from control and diabetic rats, showing a significant reduction in ROBO4 expression in diabetes (n=3 or 4, *p<0.05 vs Wistar). Scale bars, 10 μm

Article Snippet: ROBO4 overexpression In vivo experiments Robo4 adenovirus (3 μl of 1.85 × 10 12 viral particles/ml, Ad- Gfp -h Robo4 , ADV-221472; Vector Biolabs, Philadelphia, PA, USA) was injected over 6 min via a 30-gauge needle adjacent to the MCA at stereotactic coordinates (bregma: anterior–posterior, −0.5 mm; mediolateral −1 mm; dorsoventral, −4 mm).

Techniques: Expressing, Injection, Control, Western Blot, Isolation

Journal: Diabetologia

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

doi: 10.1007/s00125-017-4214-6

Figure Lengend Snippet: ROBO4 overexpression increases vascular stability. (a) Nuclear morphology was used to assess the pericyte-to-endothelial cell ratio. Dotted lines represent the elliptical endothelial cells nuclei and solid circles represent pericytes nuclei. Overexpression of ROBO4 (Robo4 AD) significantly increased this ratio in the cerebral vasculature (n=4, *p<0.05 vs contralateral side). Scale bars, 10 μm. (b) Representative images of brain sections showing FITC-filled vessels (green), nuclear stain (DAPI, blue), pericyte marker, PDGF receptor-β antibody (red) and merged images. Scale bars, 10 μm

Article Snippet: ROBO4 overexpression In vivo experiments Robo4 adenovirus (3 μl of 1.85 × 10 12 viral particles/ml, Ad- Gfp -h Robo4 , ADV-221472; Vector Biolabs, Philadelphia, PA, USA) was injected over 6 min via a 30-gauge needle adjacent to the MCA at stereotactic coordinates (bregma: anterior–posterior, −0.5 mm; mediolateral −1 mm; dorsoventral, −4 mm).

Techniques: Over Expression, Staining, Marker

Journal: Diabetologia

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

doi: 10.1007/s00125-017-4214-6

Figure Lengend Snippet: Increased VEGF signalling in cerebral vessels in diabetes decreases the expression and availability of ROBO4 via binding with VEGF-activated β3 integrin. Overexpression of ROBO4 reduces the augmented VEGF-induced angiogenic signal and decreases cerebral neovascularisation in diabetes. Inhibition of the VEGF angiogenic signal by SKLB1002, a selective VEGFR-2 antagonist, decreases ROBO4–β3 integrin interaction, increases the expression and availability of ROBO4 and prevents/repairs cerebral neovascularisation in diabetes

Article Snippet: ROBO4 overexpression In vivo experiments Robo4 adenovirus (3 μl of 1.85 × 10 12 viral particles/ml, Ad- Gfp -h Robo4 , ADV-221472; Vector Biolabs, Philadelphia, PA, USA) was injected over 6 min via a 30-gauge needle adjacent to the MCA at stereotactic coordinates (bregma: anterior–posterior, −0.5 mm; mediolateral −1 mm; dorsoventral, −4 mm).

Techniques: Expressing, Binding Assay, Over Expression, Inhibition