Notch-1 signalling is activated in brain arteriovenous malformations in humans

Abstract

A role for the Notch signalling pathway in the formation of arteriovenous malformations during development has been suggested. However, whether Notch signalling is involved in brain arteriovenous malformations in humans remains unclear. Here, we performed immunohistochemistry on surgically resected brain arteriovenous malformations and found that, compared with control brain vascular tissue, Notch-1 signalling was activated in smooth muscle and endothelial cells of the lesional tissue. Western blotting showed an activated form of Notch-1 in brain arteriovenous malformations, irrespective of clinical presentation and with or without preoperative embolization, but not in normal cerebral vessels from controls. In addition, the Notch-1 ligands Jagged-1 and Delta-like-4 and the downstream Notch-1 target Hes-1 were increased in abundance and activated in human brain arteriovenous malformations. Finally, increased angiogenesis was found in adult rats treated with a Notch-1 activator. Our findings suggest that activation of Notch-1 signalling is a phenotypic feature of brain arteriovenous malformations, and that activation of Notch-1 in normal vasculature induces a pro-angiogenic state, which may contribute to the development of vascular malformations.

Figure 1

Activation of Notch-1 signalling in human brain AVMs. (A) NICD was barely detectable in normal human cerebral vessels (Control), but was highly expressed in cell nuclei of AVMs. (B) Double immunostaining of human brain AVM shows nuclear NICD (green) and cytoplasmic SMC (red) in the same (vascular smooth muscle) cells (arrows). DAPI (blue) was used to counterstain nuclei. (C) Double immunostaining of human brain AVM shows nuclear NICD (green) and cytoplasmic CD31 (red) in the same (endothelial) cells (arrows). DAPI (blue) was used to counterstain nuclei. (D) Double immunostaining of normal human middle cerebral artery shows abundant cytoplasmic SMC (green) but little nuclear NICD (red). DAPI (blue) was used to counterstain nuclei. (E) Protein was isolated from normal human frontal cortex (H-FCX), caudate-putamen (H-Cpu) and middle cerebral artery (H-MCA) and from four human AVMs (AVM 1–4). Western blotting was performed using an antibody against NICD (top panel). A band of the predicted size (arrow) was blocked after pre-incubating anti-NICD with an excess of immunizing peptide (Peptide). The membrane was re-probed with anti-actin as an internal protein loading control (bottom panel, arrow).

Figure 2

Immunohistochemical detection of Notch-1 ligands and Hes-1 in human brain AVMs. (A) Dll4, Jagged-1 and Hes-1 expression (black) was increased in human brain AVMs (left panels, low-magnification; middle panels, high-magnification) compared with normal human middle cerebral artery (right panels). A few non-vascular cells in normal control brain also expressed Hes-1 (arrow). (B) Notch1, Jagged-1, Dll4, NICD and Hes-1 were expressed or activated in vascular cells of E17 mouse brain. (C) Immunostaining was performed on H9 human embryonic stem cells using (left to right) anti-Jagged-1 (green) and anti-NICD (red); anti-Notch-1 (green); anti-Dll4 (green) and anti-Hes1 (green). DAPI (blue) was used to counterstain nuclei.

Figure 3

Western blot analysis and cell-type localization of Notch-1 ligands in human brain AVMs. (A) Protein was isolated from the sources listed in the legend to Figure 1, and from normal mouse cerebral cortex (M-CTX). Western blotting was performed using antibodies against the Notch-1 ligands Dll4 (top panel) and Jagged-1 (middle panel), with anti-actin to control for differences in protein loading (bottom panel). (B) Recombinant human Notch-1, Jagged-1, Dll4 and Hes1 were loaded on gels and probed with the indicated antibodies, showing that the antibodies used recognize authentic antigens. Each pair of gel lanes was loaded with 100 ng (lane 1) or 500 ng (lane 2) of total protein. (C) Double-label immunostaining of human brain AVM sections shows that αSMC-expressing vascular smooth muscle cells also express Jagged-1 (top panel) and Dll4 (bottom panel). DAPI (blue) was used to counterstain nuclei. (D) Triple-label immunostaining of human brain AVM shows co-expression of Jagged-1 (red) and Hes1 (green) in CD31-immunopositive (purple) endothelial cells. DAPI (blue) was used to counterstain nuclei.

Figure 4

Effects of Notch-1 signalling activation in vivo on cerebral vessels of rat brain. (A) Vehicle (left panel) or Notch-1 activator (right panel) was infused into the lateral ventricle of adult rat brain for 7 days. BrdU was given by intraperitoneal injection for the first 3 days, and rats were killed on day 8. Notch-1-activating mouse antibody entered the brain parenchyma (right panel), as evidenced by immunostaining with a hamster anti-mouse isotypic antibody (brown). (B) Double-label immunostaining shows expression of activated Notch-1 (NICD) in lectin-labelled endothelial cells (left), αSMC-immunopositive vascular smooth muscle cells (middle) and neuron-specific nuclear protein (NeuN)-expressing neurons of Notch-1 activator-treated brain AVM (top panel) but not in vehicle-treated control brain (bottom panel). Insert in top left panel shows NICD immunoreactivity at higher magnification. DAPI (blue) was used to counterstain nuclei. (C) BrdU-positive cells were found in cerebrovascular endothelial (red arrows) and smooth muscle (white arrows) cells of Notch-1 activator-treated (right and middle top panels), but not control (middle lower panel) rat brains. (D) Double-label staining of sections from rat brain after administration of Notch-1 activator shows BrdU-immunopositive nuclei in lectin-labelled (vascular endothelial) cells. (E) Double-label immunostaining of rat brain vessels after administration of Notch-1 activator also shows BrdU-positive nuclei (red) in αSMC-expressing (green) smooth muscle cells. DAPI (blue) was used to counterstain nuclei. (F) The number of BrdU-positive cells per 400× field in rat cortex was increased by administration of Notch-1 activator, compared with Notch-1 inhibitor or vehicle. Asterisk indicates P < 0.05 relative to vehicle treatment (ANOVA and Bonferroni post hoc tests).

Figure 5

Effect of Notch-1 activator on rat brain blood vessels. (A) The number of FITC-lectin-positive cerebral cortical blood vessels was increased after 7 days in the Notch-1 activator- (left panel) compared with the Notch-1 inhibitor- (middle panel) and vehicle-treated control (right panel) groups. FITC-lectin-labelled blood vessels were counted manually (B) and the area (C) and intensity (D) of FITC-lectin staining was measured using IMARIS software, in cerebral cortex of rats given a Notch-1 activator, Notch-1 inhibitor or vehicle for 7 days. (E) Notch-1 activator was continuously infused into the lateral ventricle of adult rat brain for 28 days, and rats were killed 1 day later. Immunostaining with anti-αSMC (brown) shows that compared with vehicle-treated controls (left), cerebral vessels from rats given a Notch-1 activator (right) have thicker vessel walls. Asterisks indicate P < 0.05 (ANOVA and Bonferroni post hoc tests) relative to vehicle-treated controls.