Endorepellin downregulation promotes angiogenesis after experimental traumatic brain injury

Figures

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  Figure 1. Experimental design and validation of effectiveness of adeno-associated virus transfection. Mice were randomly divided into four groups: sham, controlled cortical impact (CCI), AAV-green fluorescent protein (GFP), and AAV-shEndorepellin-GFP. For the in vitro study, cells were divided into four groups: sham, control (stretch injury only), AAV-GFP, and AAV-shEndorepellin-GFP. (A) Graph illustrating the experimental design including traumatic brain injury, AAV transfection, and neurobehavioral assessments. (B) Representative western blot bands and quantification of endorepellin protein expression in human umbilical vein endothelial cells harvested 72 hours after AAV-shEndorepellin-GFP transfection. n = 5 per group. (C) Representative western blot bands and quantification of endorepellin protein expression in the cortex of mice at 2 weeks after AAV vector injection. n = 5 per group. Data are expressed as mean ± SD. **P < 0.01 (one-way analysis of variance followed by Bonferroni’s post hoc test). AAV-shEndorepellin-GFP is abbreviated as AAV-shEp-GFP in the figure. AAV-shEndorepellin-GFP abbreviates as AAV-shEp-GFP in the figure. AAV: Adeno-associated virus; CCI: controlled cortical…

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  Figure 2. Downregulation of endorepellin improves neurobehavioral recovery in CCI mice. (A) Downregulation of endorepellin resulted in significant improvement of modified neurological severity scores (mNSS) on days 7 and 14 post-CCI, compared with the AAV-GFP-treated group. (B) Downregulation of endorepellin increased the time spent on the rotarod on days 7 and 14 post-CCI, compared with the AAV-GFP-treated group. n = 6 per group. Data are expressed as mean ± SD. *P < 0.05, AAV-shEndorepellin-GFP, vs. CCI group (one-way analysis of variance followed by Bonferroni’s post hoc test). AAV-shEndorepellin-GFP abbreviates as AAV-shEp-GFP in the figure. AAV: Adeno-associated virus; CCI: controlled cortical impact; GFP: green fluorescent protein.

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  Figure 3. AAV-shEndorepellin-GFP brain transduction increases perfused small vessel density in CCI mice. (A) Schematic diagram of immunostained images. Black boxes represent the areas of each section selected for imaging, and the gray area represents the lesion brain region. (B) Representative images showing immunostaining of CD31+ microvessels in the ipsilateral hemisphere after controlled cortical impact (CCI) in the sham, CCI, GFP, and AAV-shEndorepellin-GFP treatment groups. Scale bar: 100 μm, n = 6 per group. (C) Synchrotron radiation angiography showed microvessels in the peri-lesional area at 14 days after CCI. Black boxes show the areas used for measurement. Scale bar: 500 μm, n = 5 per group. (D, E) Bar graphs show the length and diameter of vessels in the ipsilateral hemisphere of CCI mice at 2 weeks after AAV injection. n = 5 per group. *P < 0.05, ***P < 0.001, vs. CCI group (one-way analysis of variance followed by Bonferroni’s post hoc test). (F) Bar graphs show perfused vessel density in sham, GFP and AAV-shEndorepellin-GFP treatment groups 14 days after CCI. **P < 0.01, vs. AAV-GFP group (one-way analysis of variance followed by Bonferroni’s post hoc test). Data are…

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  Figure 4. AAV-shEndorepellin-GFP brain transduction promotes angiogenesis 14 days after CCI. (A) Representative images showing immunostaining of CD31 (red) and Ki-67 (green) double-stained cells in CCI mice. Scale bar: 100 μm, n = 6 per group. (B) Bar graphs show quantification of CD31+/Ki-67+ cells in the AAV-shEndorepellin-GFP, AAV-GFP, CCI, and sham groups. (C) Representative western blots showing vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) expression in the brain 14 days after CCI. n = 5 per group. (D, E) Bar graphs showing quantification of Ang-1 and VEGF protein expression. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, AAV-shEndorepellin-GFP vs. CCI group (one-way analysis of variance followed by Bonferroni’s post hoc test). AAV-shEndorepellin-GFP abbreviates as AAV-shEp-GFP in the figure. AAV: Adeno-associated virus; CCI: controlled cortical impact; DAPI: 4′,6-diamidino-2-phenylindole; GFP: green fluorescent protein.

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  Figure 5. Endorepellin downregulation improves tube formation of human umbilical vein endothelial cells. Representative images and bar graph show tube formation ability of human umbilical vein endothelial cells after transduction with AAV-shEndorepellin-GFP. Scale bar: 100 μm, n = 5 per group. Data are expressed as mean ± SD. **P < 0.01 (one-way analysis of variance followed by Bonferroni’s post hoc test). AAV-shEndorepellin-GFP abbreviates as AAV-shEp-GFP in the figure. AAV-shEndorepellin-GFP abbreviates as AAV-shEp-GFP in the figure. AAV: Adeno-associated virus; GFP: green fluorescent protein.

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  Figure 6. Endorepellin downregulation increased Ang-1, Ang-2, VEGF, and EGFR expression in human umbilical vein endothelial cells after mechanical stretch injury. (A–E) Representative western blot bands (A) and quantification (B–E) of Ang-1, Ang-2, VEGF, and EGFR protein expression at 24 hour after injury in different groups. n = 6 per group. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, vs. AAV-GFP group (one-way analysis of variance followed by Bonferroni’s post hoc test). AAV-shEndorepellin-GFP abbreviates as AAV-shEp-GFP in the figure. AAV: Adeno-associated virus; Ang-1: angiopoietin-1; Ang-2: angiopoietin-2; EGFR: epidermal growth factor receptor; GFP: green fluorescent protein; VEGF: vascular endothelial growth factor.