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. 2011 Dec;31(12):2343-51.
doi: 10.1038/jcbfm.2011.97. Epub 2011 Jul 20.

Coexpression of angiopoietin-1 with VEGF increases the structural integrity of the blood-brain barrier and reduces atrophy volume

Fanxia Shen  1 , Espen J Walker, Lidan Jiang, Vincent Degos, Jianping Li, Baoliang Sun, Fransisca Heriyanto, William L Young, Hua Su
Affiliations

Coexpression of angiopoietin-1 with VEGF increases the structural integrity of the blood-brain barrier and reduces atrophy volume

Fanxia Shen et al. J Cereb Blood Flow Metab. 2011 Dec.

Abstract

Vascular endothelial growth factor (VEGF)-induced neovasculature is immature and leaky. We tested if coexpression of angiopoietin-1 (ANG1) with VEGF improves blood-brain barrier (BBB) integrity and VEGF neuroprotective and neurorestorative effects using a permanent distal middle cerebral artery occlusion (pMCAO) model. Adult CD-1 mice were injected with 2 × 10(9) virus genomes of adeno-associated viral vectors expressing VEGF (AAV-VEGF) or ANG1 (AAV-ANG1) individually or together in a 1:1 ratio into the ischemic penumbra 1 hour after pMCAO. AAV-LacZ was used as vector control. Samples were collected 3 weeks later. Compared with AAV-LacZ, coinjection of AAV-VEGF and AAV-ANG1 reduced atrophy volume (46%, P=0.004); injection of AAV-VEGF or AAV-ANG1 individually reduced atrophy volume slightly (36%, P=0.08 and 33%, P=0.09, respectively). Overexpression of VEGF reduced tight junction protein expression and increased Evans blue extravasation. Compared with VEGF expression alone, coexpression of ANG1 with VEGF resulted in upregulation of tight junction protein expression and reduction of Evans blue leakage (AAV-ANG1/AAV-VEGF: 1.4 ± 0.3 versus AAV-VEGF: 2.8 ± 0.7, P=0.001). Coinjection of AAV-VEGF and AAV-ANG1 induced a similar degree of angiogenesis as injection of AAV-VEGF alone (P=0.85). Thus, coexpression of ANG1 with VEGF improved BBB integrity and resulted in better neuroprotection compared with VEGF expression alone.

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Figures

Figure 1
Figure 1
Schematic drawing showing the experimental design. Adeno-associated viral vectors (AAVs) were injected into two sites (red arrows) at the ischemic penumbra 1 hour after middle cerebral artery occlusion (MCAO). Dark blue highlights the infarct area and light blue highlights the penumbra. Brain samples were collected 3 weeks after vector injection. The squares show the regions used for histological analysis. The color reproduction of this figure is available at online version.
Figure 2
Figure 2
Adeno-associated viral vector (AAV) mediated transgene expression. (A) A representative image of a coronal section shows LacZ expression around the injection sites at the border of the infarct area. (B) Representative images of vascular endothelial growth factor (VEGF) and angiopoietin-1 (ANG1) antibody-stained sections. VEGF expression was detected in the VEGF and VEGF/ANG1 groups (brown). ANG1 expression was detected in the ANG1 and VEGF/ANG1 groups (red). Scale bars are 100 μm. (C) Representative Western blots are shown. β-Actin was used as loading control. (D) Bar graph shows the quantification of the protein expression analyzed from Western blots. The expression of VEGF and ANG1 increased only in corresponding vector-injected brain tissues. *P<0.05 versus AAV-LacZ group, N=3 per group. LacZ: AAV-LacZ-injected group; ANG1: AAV-ANG1-injected group; VEGF: AAV-VEGF-injected group; and VEGF/ANG1: AAV-VEGF and AAV-ANG1-coinjected groups. The color reproduction of this figure is available at online version.
Figure 3
Figure 3
Coinjection of AAV-ANG1 and AAV-VEGF reduced atrophy volume. (A) Cresyl violet-stained sections are shown. Dotted lines outline the original brain size before atrophy of the infarct regions. (B) Bar graph shows the quantification of the atrophy volume; N=6. Compared with AAV-LacZ, injection of AAV-VEGF or AAV-ANG1 resulted in a trend toward reduction of infarct (P=0.08; P=0.09); coinjection of AAV-VEGF with AAV-ANG1 reduced the atrophy volume significantly compared with the AAV-LacZ and AAV-ANG1 groups (*P=0.004 versus AAV-LacZ group; #P=0.03 versus AAV-ANG1 group). LacZ: AAV-LacZ-injected group; ANG1: AAV-ANG1-injected group; VEGF: AAV-VEGF-injected group; and VEGF/ANG1: AAV-VEGF and AAV-ANG1-coinjected groups.
Figure 4
Figure 4
Expression of tight junction proteins decreased in the AAV-VEGF-injected brain and was restored in the AAV-VEGF/AAV-ANG1-coinjected brain. (A) Representative images show tight junction protein expression. Vessels were visualized by immunolabeling endothelial cells with anti-CD31 antibody (green). ZO-1, occludin, and claudin-5 were detected by antibodies specific to the proteins (red). Arrows indicated the positions that were selected to show colocalization of endothelial cells and tight junction proteins. Scale bar=100 μm. (B) Representative images of Western blots are shown. β-Actin was used as loading control. (C) Bar graph shows quantification of the Western blot analysis. *P<0.05 versus AAV-VEGF-injected group. LacZ: AAV-LacZ-injected group; ANG1: AAV-ANG1-injected group; VEGF: AAV-VEGF-injected group; and VEGF/ANG1: AAV-VEGF and AAV-ANG1-coinjected groups. The color reproduction of this figure is available at online version.
Figure 5
Figure 5
Coexpression of angiopoietin-1 (ANG1) with vascular endothelial growth factor (VEGF) reduced blood–brain barrier (BBB) leakage caused by VEGF overexpression. (A) Images of the brain perfused with Evans blue are shown. More blue color was observed in the infarct region of the AAV-VEGF-injected group than other groups. (B) Bar graph shows the quantification of Evans blue extravasation; N=6. *P<0.01, versus AAV-LacZ- and AAV-ANG1-treated mice. Coinjection of AAV-VEGF and AAV-ANG1 reduced Evans blue extravasation compared with injection of AAV-VEGF alone (P=0.001). LacZ: AAV-LacZ-injected group; ANG1: AAV-ANG1-injected group; VEGF: AAV-VEGF-injected group; and VEGF/ANG1: AAV-VEGF and AAV-ANG1-coinjected groups. The color reproduction of this figure is available at online version.
Figure 6
Figure 6
Injection of AAV-VEGF, AAV-ANG1, or AAV-VEGF/AAV-ANG1 induced angiogenesis. (A) Representative images of CD31 and Ki67 antibody-stained sections are shown. There were more vessels and CD31/Ki67 double positive cells in the AAV-VEGF, AAV-ANG1, or AAV-VEGF/AAV-ANG1-injected brain than in the AAV-LacZ-injected brain. Arrows indicate cells that were selected to show colocalization of endothelial cells and Ki67-positive nuclei. Scale bars=100 μm. (B) Bar graph shows the quantification of vascular density. *P<0.05 versus LacZ group. **P⩽0.05 versus AAV-ANG1 group. (C) Bar graph shows the quantification of Ki67-positive endothelial cells. *P<0.05, versus LacZ group. N=7 for each group. LacZ: AAV-LacZ-injected group; ANG1: AAV-ANG1-injected group; VEGF: AAV-VEGF-injected group; and VEGF/ANG1: AAV-VEGF and AAV-ANG1-coinjected groups. The color reproduction of this figure is available at online version.
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