Endothelial Notch signaling is upregulated in human brain arteriovenous malformations and a mouse model of the disease

Abstract

Brain arteriovenous malformations (BAVMs) can cause lethal hemorrhagic stroke and have no effective treatment. The cellular and molecular basis for this disease is largely unknown. We have previously shown that expression of constitutively-active Notch4 receptor in the endothelium elicits and maintains the hallmarks of BAVM in mice, thus establishing a mouse model of the disease. Our work suggested that Notch pathway could be a critical molecular mediator of BAVM pathogenesis. Here, we investigated the hypothesis that upregulated Notch activation contributes to the pathogenesis of human BAVM. We examined the expression of the canonical Notch downstream target Hes1 in the endothelium of human BAVMs by immunofluorescence, and showed increased levels relative to either autopsy or surgical biopsy controls. We then analyzed receptor activity using an antibody to the activated form of the Notch1 receptor, and found increased levels of activity. These findings suggest that Notch activation may promote the development and even maintenance of BAVM. We also detected increases in Hes1 and activated Notch1 expression in our mouse model of BAVM induced by constitutively active Notch4, demonstrating molecular similarity between the mouse model and the human disease. Our work suggests that activation of Notch signaling is an important molecular candidate in BAVM pathogenesis and further validates that our animal model provides a platform to study the progression as well as the regression of the disease.

Figure 1. Hes1 staining of crypt cell nuclei of the gut confirms antibody specificity

(a–b) Hes1 antibody positive control. Typical staining pattern shown by immunofluorescence in the crypt cells of mouse large (a) and small intestine (b), demonstrates specificity of the Hes1 antibody. High magnification of the crypt (boxed area in b) shows co-localization of Hes1 and DAPI nuclear label (b-1 to b-2). (c–d) Negative control for Hes1 staining, non-specific IgG in adjacent sections. (e) Typical staining pattern shown by immunofluorescence in the crypt cells of human small intestine, demonstrates the specificity of the Hes1 antibody. High magnification confocal imaging of the crypt shows co-localization of Hes1 and DAPI nuclear label (e-1 to e-3). (f) Negative control with non-specific IgG in an adjacent section to (e) does not show the same staining pattern. Scale bars (a&c) 100 μm, (b&d) 50 μm, (e&f) 100 μm.

Figure 2. Increased Hes1 staining in the endothelium of human brain arteriovenous malformations (BAVM)s

(a–b) Immunofluorescent staining for Hes1 in paraffin sections from surgical biopsy controls (a) and human BAVMs (b). Increased Hes1 staining is evident in the endothelium of human BAVMs (arrowheads, b). (c) Negative control for Hes1 staining, non-specific IgG in an adjacent section to (b). (d&e) CD31 stained sections adjacent to (a&b), respectively. Low-magnification (e′) shows the location of (e) within a large vascular structure (box with asterisk). High-magnification of the boxed areas in (a–d) shows co-localization of Hes1 staining with the DAPI nuclear label (a-1 to c-1 and a-2 to c-2) in cells lining CD31 labeled vessels (d-1&e-1). (f) Quantification of Hes1 staining in BAVM cases and autopsy or surgical biopsy controls. Scale bars (a–e) 200 μm. In graph (N=12 BAVM samples, N=7 autopsy controls, N=4 biopsy controls; BAVM vs. autopsy controls P=0.0003; BAVM vs. biopsy controls P=0.05). Average represents mean ± SEM of cases. Individual cases represent mean ± SEM of individual endothelial cells in each case.

Figure 3. Increased activated-Notch1 staining in the endothelium in human brain arteriovenous malformations (BAVM)s

(a–b) Immunofluorescent staining for activated-Notch1 in paraffin sections from surgical biopsy controls (a) and human BAVMs (b). Increased activated-Notch1 staining is evident in the endothelium of human BAVMs (arrowheads, b). (c) Negative control for activated-Notch1 antibody staining, non-specific IgG in an adjacent section to (b). (d&e) CD31 stained adjacent sections to (a) and (b). Low-magnification (e′) shows the location of the stained area within a large vascular structure (box with asterisk). (f) High-magnification of the boxed areas shows co-localization of activated-Notch1 staining with the DAPI nuclear label (a-1 to c-1 and a-2 to c-2) in cells lining the CD31 labeled vessels (d-1 & e-1). (f) Quantification of activated-Notch1 staining in BAVM cases and autopsy or surgical biopsy controls (N=14 BAVM samples, N=7 autopsy controls, N=4 biopsy controls; BAVM vs. autopsy controls P=0.0001; BAVM vs. biopsy controls P=0.008). Scale bar is 200 μm. Average represents mean ± SEM of cases. Individual cases represent mean ± SEM of individual endothelial cells in each case.

Figure 4. Increased Hes1 staining in the endothelium of the Notch4*-Tet mouse model of brain arteriovenous malformation (BAVM)-like lesions

(a–b) Immunofluorescent staining for Hes1 in paraffin sections from Notch4*-Tet mice (a) and genetic controls (b). Increased Hes1 staining is evident in the endothelium of FITC-lectin perfused vessels in Notch4*-Tet mice. (c) Negative control for Hes1 staining, non-specific IgG in an adjacent section to (a). High-magnification of the boxed areas shows co-localization of Hes1 staining with the DAPI nuclear label (a-1 to c-1 and a-2 to c-2) in cells lining the lectin perfused vessel lumen (a-3 to c-3). (d) Quantification of Hes1 staining relative to non-specific IgG staining in Notch4* expressing mutant mice and controls (N=5 genetic controls, N=3 Notch4*-Tet mutants, values represent mean ± SEM, P=0.0004). Scale bars (a–c) 200 μm.

Figure 5. Increased activated-Notch1 staining in the endothelium of the Notch4*-Tet mouse model of brain arteriovenous malformation (BAVM)-like lesions

(a–c) Immunofluorescent staining for activated-Notch1 in paraffin sections from Notch4*-Tet mice (a) and genetic controls (b). Increased activated-Notch1 staining is evident in the endothelium of FITC-lectin perfused vessels in Notch4*-Tet mice (a). (c) Negative control for activated-Notch1 staining, non-specific IgG in an adjacent section to (a). High-magnification of the boxed areas shows co-localization of activated-Notch1 staining with the DAPI nuclear label (a-1 to c-1 and a-2 to c-2) in cells lining the lectin perfused vessel lumen (a-3 to c-3). (d) Quantification of activated-Notch1 staining relative to non-specific IgG staining in Notch4* expressing mutant mice and controls. (N=5 genetic controls, N=3 Notch4*-Tet mutants, values represent mean ± SEM, P=0.004). Scale bar is 100 μm.