Notch receptor expression in human brain arteriovenous malformations

Abstract

The roles of the Notch pathway proteins in normal adult vascular physiology and the pathogenesis of brain arteriovenous malformations are not well-understood. Notch 1 and 4 have been detected in human and mutant mice vascular malformations respectively. Although mutations in the human Notch 3 gene caused a genetic form of vascular stroke and dementia, its role in arteriovenous malformations development has been unknown. In this study, we performed immunohistochemistry screening on tissue microarrays containing eight surgically resected human brain arteriovenous malformations and 10 control surgical epilepsy samples. The tissue microarrays were evaluated for Notch 1-4 expression. We have found that compared to normal brain vascular tissue Notch-3 was dramatically increased in brain arteriovenous malformations. Similarly, Notch 4 labelling was also increased in vascular malformations and was confirmed by western blot analysis. Notch 2 was not detectable in any of the human vessels analysed. Using both immunohistochemistry on microarrays and western blot analysis, we have found that Notch-1 expression was detectable in control vessels, and discovered a significant decrease of Notch 1 expression in vascular malformations. We have demonstrated that Notch 3 and 4, and not Notch 1, were highly increased in human arteriovenous malformations. Our findings suggested that Notch 4, and more importantly, Notch 3, may play a role in the development and pathobiology of human arteriovenous malformations.

Keywords: BAVMs; cell signalling; endothelial cells; vascular malformations.

Figure 1

Haematoxylin and Eosin staining of control (A and C) and AVM blood vessels (B and D). Elastin Antibody labelling in control (E) and AVM tissue (F). Arrows show areas of elastin antibody labelling. 200× (A and B), 400× (C–F).

Figure 2

Immunohistochemistry for Notch 1 in control (A and C). Arrows indicate Notch 1 present in the interior wall of the blood vessels of the control sections. Arrow head indicates a blood vessel with minimal Notch 1 labelling. BAVM TMA samples (B and D). Arrows indicate vessels with decrease Notch 1 labelling in BAVMs. 200× (A and B), 400× (C and D).

Figure 3

Western Blot for Notch 1 in control (C1–3) and AVM (A1–3) fresh surgical samples. Multiple bands could be identified between 76–150 MW. The bracket indicates the bands that were included in the quantification. Bar graphs represent Total Density after they were normalized with the B-actin internal control antibody. Graphs were made using Graph Pad Prism 5 Software, ***P = 0.0007.

Figure 4

Immunohistochemistry for Notch 4 protein in control (A and C) and co-labelled with elastin (E). Arrow indicates the elastin staining in the IEL of the blood vessel shown. Immunohistochemistry for Notch 4 protein in BAVM tissue (B and D) and co-labelled with elastin (F). Arrow head indicates elastin staining. 200× (A and B), 400× (C–F).

Figure 5

Western Blot for Notch 4 protein in AVM (A1–4) and control (C1–3) fresh surgical samples. A single band at molecular weight 53 could be detected for Notch 4. Bar graphs represent Total Density after they were normalized with the B-actin internal control antibody. Graphs were made using Graph Pad Prism 5 Software, **P = 0.0030.

Figure 6

Immunohistochemistry for Notch 3 in control epilepsy (A and C) and co-labelled with elastin (E). BAVM tissue (B and D) and co-labelled with elastin (F). Arrows indicate Notch 3 labelling on blood vessel walls. Arrow head indicates elastin staining in the IEL of control vessels. 200× (A and B), 400× (C–F).