Endothelial Notch4 signaling induces hallmarks of brain arteriovenous malformations in mice

Abstract

Brain arteriovenous malformations (BAVMs) can cause devastating stroke in young people and contribute to half of all hemorrhagic stroke in children. Unfortunately, the pathogenesis of BAVMs is unknown. In this article we show that activation of Notch signaling in the endothelium during brain development causes BAVM in mice. We turned on constitutively active Notch4 (int3) expression in endothelial cells from birth by using the tetracycline-regulatable system. All mutants developed hallmarks of BAVMs, including cerebral arteriovenous shunting and vessel enlargement, by 3 weeks of age and died by 5 weeks of age. Twenty-five percent of the mutants showed signs of neurological dysfunction, including ataxia and seizure. Affected mice exhibited hemorrhage and neuronal cell death within the cerebral cortex and cerebellum. Strikingly, int3 repression resolved ataxia and reversed the disease progression, demonstrating that int3 is not only sufficient to induce, but also required to sustain the disease. We show that int3 expression results in widespread enlargement of the microvasculature, which coincided with a reduction in capillary density, linking vessel enlargement to Notch's known function of inhibiting vessel sprouting. Our data suggest that the Notch pathway is a molecular regulator of BAVM pathogenesis in mice, and offer hope that their regression might be possible by targeting the causal molecular lesion.

Fig. 1.

Endothelial expression of int3 causes ataxia and death in neonatal mice. (A and B) Increased Notch4 activation specifically in the ECs lining lectin-perfused vessels of P29 mutant brain was revealed by nuclear staining of Notch4 intracellular domain (N4-ICD). DAPI-labeled EC nuclei (white arrowheads). (C) Kaplan–Meier survival curve shows that all mutants died by P36. (Scale bar, 50 μm.)

Fig. 2.

Brain hemorrhage occurred in all mutant mice. (A and B) Multifocal hemorrhage (arrowheads) revealed in mutants after vascular perfusion. (C and D) H&E stained sagittal sections of perfusion fixed cerebellum from a mutant with ataxia show hemorrhage (green asterisk) and thrombosis (green arrowhead). (E) H&E stained sagittal section of cerebellar folia from a mutant without ataxia shows hemorrhage (green asterisk) and adjacent dropout of granular and Purkinje neurons (yellow arrowhead). Normal neuronal architecture (yellow arrow) is away from the hemorrhage. (F) H&E stained axial section of a severely affected mutant shows intraventricular hemorrhage (white arrow) immediately adjacent to a large, thrombosed vessel (green arrowhead) and parenchymal hemorrhage (green asterisk) in the cerebrum. (Scale bars: C and F, 400 μm; D, 100 μm; E, 200 μm.)

Fig. 3.

Enlarged and tangled blood vessels developed in all mutant mice. Vessel enlargement occurred within the parenchyma and on the surface of the brain. Large tangled vessels were shown in the parenchyma of the cerebellum and midbrain by vascular casting at P27 (B; white arrowheads) and in the lateral meninges by whole mount imaging of Cy3-labeled lectin perfusion (D). (A and C) Abnormal vessels were not observed in littermate controls. (Scale bar, 200 μm.)

Fig. 4.

Increased vessel size and decreased vessel density correlated with the frequency of hemorrhage. Shown is immunofluorescence in sagittal sections of brain regions. Density graphs represent FITC-lectin perfused (red) and total CD31 immunostained (black) vessels in each region (per squared millimeter). Δ Proportion of vessel with given diameter represents the percent change in the proportion of small (<7.5 μm), medium (7.5–20 μm), and large (>20 μm) diameter vessels in each region, relative to average controls. (A and B) Tie2-tTA (n = 6) and Tie2-tTA;TRE-int3 (n = 3); (C, D, E, and F), Tie2-tTA (n = 6) and Tie2-tTA;TRE-int3 (n = 5). Values represent mean ± SEM. (Scale bars, 200 μm.)

Fig. 5.

All mutants developed shunting and arteriovenous malformations. (A) Increased carotid blood flow was detected by P21. Maximal (systolic) carotid blood velocity was measured by pulsed-wave Doppler ultrasound. Tie2-tTA and Tie2-tTA;TRE-int3 (n = 9 and n = 6) at P15; (n = 12 and n = 14) at P17; (n = 16 and n = 26) at P21; (n = 18 and n = 12) at P23; (n = 14 and n = 9) at P29; values represent mean ± SEM. Changes were highly significant from P21 (***, P < 0.00005). Typical Doppler traces from mice at P21 (A Right). (B) Brain arteriovenous shunts developed by P19 as shown by fluorescent microsphere passage. The microspheres bypassed the brain and lodged in the lung in the mutants but not controls. BF, Bright-field; FITC, green fluorescent images.

Fig. 6.

Enlarged arteriovenous connections developed on the meningial surface and in the parenchyma of mutant brains. (A and B) X-gal staining of ephrin-B2^+/tLacZ brains revealed the middle cerebral artery (MCA, red arrowheads) on the lateral side of the brain. High magnification image of the boxed area in B shows direct arteriovenous connections (black arrowheads) between arterial branches of the MCA (A) and venous vessels (V) in Tie2-tTA;TRE-int3;ephrin-B2^+/tLacZ mice (B′). (C and D) Fluorescent images of sagittal cerebellar 100-μm thick sections show enlarged connections (white arrowheads) between GFP-labeled interfolial arteries of Tie2-tTA;TRE-int3;ephrin-B2^+/tLacZ mice (A) and draining veins (V) in the mutant (D) and not control (C). (Scale bar, 50 μm.)

Fig. 7.

Repression of int3 resolved ataxia and prevented death. (A) Repression of int3 with Dox at P20 or P21 allowed the survival of mutant mice, as shown by the Kaplan–Meier curve. Tie2-tTA;TRE-int3 Dox-off (n = 23) and Tie2-tTA;TRE-int3 Dox-on (n = 15) at 20 days; Tie2-tTA;TRE-int3 Dox-off (n = 0) and Tie2-tTA;TRE-int3 Dox-on (n = 5) at 40 days. (B) Ataxia was resolved by int3 repression. Still frames were taken from a movie of a severely ataxic mutant at P19, and upon its recovery at P22, after 3 days of Dox treatment.