Effect of elevation of vascular endothelial growth factor level on exacerbation of hemorrhage in mouse brain arteriovenous malformation

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OBJECTIVE

A high level of vascular endothelial growth factor (VEGF) has been implicated in brain arteriovenous malformation (bAVM) bleeding and rupture. However, direct evidence is missing. In this study the authors used a mouse bAVM model to test the hypothesis that elevation of focal VEGF levels in bAVMs exacerbates the severity of bAVM hemorrhage.

METHODS

Brain AVMs were induced in adult mice in which activin receptor–like kinase 1 (Alk1, a gene that causes AVM) gene exons 4–6 were floxed by intrabasal ganglia injection of an adenoviral vector expressing Cre recombinase to induce Alk1 mutation and an adeno-associated viral vector expressing human VEGF (AAV-VEGF) to induce angiogenesis. Two doses of AAV-VEGF (5 × 109 [high] or 2 × 109 [low]) viral genomes were used. In addition, the common carotid artery and external jugular vein were anastomosed in a group of mice treated with low-dose AAV-VEGF 6 weeks after the model induction to induce cerebral venous hypertension (VH), because VH increases the VEGF level in the brain. Brain samples were collected 8 weeks after the model induction. Hemorrhages in the bAVM lesions were quantified on brain sections stained with Prussian blue, which detects iron deposition. VEGF levels were quantified in bAVM tissue by enzyme-linked immunosorbent assay.

RESULTS

Compared to mice injected with a low dose of AAV-VEGF, the mice injected with a high dose had higher levels of VEGF (p = 0.003) and larger Prussian blue–positive areas in the bAVM lesion at 8 or 9 weeks after model induction (p = 0.002). VH increased bAVM hemorrhage in the low-dose AAV-VEGF group. The overall mortality in the high-dose AAV-VEGF group was 26.7%, whereas no mouse died in the low-dose AAV-VEGF group without VH. In contrast, VH caused a mortality of 50% in the low-dose AAV-VEGF group.

CONCLUSIONS

Using mouse bAVM models, the authors provided direct evidence that elevation of the VEGF level increases bAVM hemorrhage and mouse mortality.

ABBREVIATIONS AAV = adeno-associated viral vector; Ad-Cre = adenoviral vector expressing Cre recombinase; Ad-GFP = adenoviral vector expressing green fluorescent protein; bAVM = brain arteriovenous malformation; CCA = common carotid artery; EJV = external jugular vein; IACUC = Institutional Animal Care and Use Committee; UCSF = University of California, San Francisco; VEGF = vascular endothelial growth factor; VH = venous hypertension; WT = wild type.

Article Information

Correspondence Hua Su: University of California, San Francisco, CA. hua.su@ucsf.edu.

INCLUDE WHEN CITING Published online April 26, 2019; DOI: 10.3171/2019.1.JNS183112.

P.C. and L.M. contributed equally to this work.

Disclosures The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.

© AANS, except where prohibited by US copyright law.

Headings

Figures

  • View in gallery

    An increase of AAV-VEGF dose increased VEGF protein in the brain. Control, uninjected brain; Low, mice injected with low-dose AAV-VEGF; High, mice injected with high-dose AAV-VEGF. There were 4 mice in the control group, 9 in the low-dose AAV-VEGF group, and 10 in the high-dose AAV-VEGF group.

  • View in gallery

    Vascular structures were severely damaged near the needle track in mice that received high-dose AAV-VEGF. A: Representative images of brain sections near the needle track in the high-dose AAV-VEGF group. Vessels are shown in white. B: Representative images of brain sections used for vessel quantifications. Dysplastic vessels are indicated by asterisks. Bar = 50 μm. C: Quantification of vessel density. D: Quantification of dysplasia index. There were 10 mice in the low-dose AAV-VEGF group and 9 in the high-dose AAV-VEGF group.

  • View in gallery

    Elevation of the AAV-VEGF dose increased bAVM hemorrhage. A: Representative images of Prussian blue–stained sections. The iron depositions (blue) in bAVMs indicate hemorrhage. The nuclei were counterstained with Fast Red. Bar = 1 mm. B: Quantification of Prussian blue–positive area. Log10: the data were 10 log-transformed. Numbers in subgroups were as follows: n = 9 for low-dose AAV-VEGF group and n = 20 for high-dose VEGF group. Figure is available in color online only.

  • View in gallery

    Brain VH exacerbated hemorrhage in bAVMs. A: Representative images of Prussian blue–stained sections. Iron depositions (blue) in bAVMs indicate hemorrhage. The nuclei were counterstained with Fast Red. Bar = 100 μm. B: Quantification of Prussian blue–positive area. Numbers in subgroups were as follows: n = 6 for CV no VH group; n = 3 for CV VH group; n = 5 for GV VH group; and n = 4 for CL VH group. CL = mice injected with Ad-GFP and AAV-LacZ (WT brain); CV = mice injected with Ad-Cre and low-dose AAV-VEGF (AVM model); GV = mice injected with Ad-GFP and low-dose AAV-VEGF (angiogenesis in WT brain). Figure is available in color online only.

  • View in gallery

    Survival curve. The 0 on the x-axis means the first day of week 7 after model induction. Numbers in subgroups were as follows: n = 15 for high-dose VEGF group; n = 11 for low-dose VEGF group; n = 6 for VH CV group; n = 6 for VH GV group; and n = 5 for VH CL group. CL = mice injected with Ad-GFP and AAV-LacZ (WT brain); CV = mice injected with Ad-Cre and low-dose AAV-VEGF (AVM model); GV = mice injected with Ad-GFP and low-dose AAV-VEGF (angiogenesis in WT brain).

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