Notch4 normalization reduces blood vessel size in arteriovenous malformations - PubMed (original) (raw)

Notch4 normalization reduces blood vessel size in arteriovenous malformations

Patrick A Murphy et al. Sci Transl Med. 2012.

Abstract

Abnormally enlarged blood vessels underlie many life-threatening disorders including arteriovenous (AV) malformations (AVMs). The core defect in AVMs is high-flow AV shunts, which connect arteries directly to veins, "stealing" blood from capillaries. Here, we studied mouse brain AV shunts caused by up-regulation of Notch signaling in endothelial cells (ECs) through transgenic expression of constitutively active Notch4 (Notch4*). Using four-dimensional two-photon imaging through a cranial window, we found that normalizing Notch signaling by repressing Notch4* expression converted large-caliber, high-flow AV shunts to capillary-like vessels. The structural regression of the high-flow AV shunts returned blood to capillaries, thus reversing tissue hypoxia. This regression was initiated by vessel narrowing without the loss of ECs and required restoration of EphB4 receptor expression by venous ECs. Normalization of Notch signaling resulting in regression of high-flow AV shunts, and a return to normal blood flow suggests that targeting the Notch pathway may be useful therapeutically for treating diseases such as AVMs.

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Figures

Figure 1

Figure 1. Repression of Notch4* induces the normalization of arteriovenous malformation

(A–H) Two-photon timelapse imaging of cortical brain vessels through a cranial window in Notch4* mutant mice. Vessel topology was visualized by intravenous FITC-dextran. Arteriovenous shunt (A&B) was reduced in diameter following the repression of Notch4* (C–H). Centerline velocity in the regressing AV shunt was obtained by direct measurement of the velocity of individual red blood cells (panels B,D,F,H). Repression of Notch4* decreased blood flow velocity in shunt by 48hrs (compare B to D). (I) Quantification of the changes in shunt diameter without repression of Notch4* (Notch4*-On) or with repression of Notch4* (Notch4*-Off) for 48hrs. Diameter was measured at the narrowest point between artery and vein in Notch4*-On mice before and after treatment (_n_=22 AV shunts in 10 mice with and n =35 AV shunts in 11 mice without Notch4* repression). Error bars represent s.e.m. between individual AV shunts. Scale bars = 50μm.

Figure 2

Figure 2. Flow analysis indicates that AVM narrowing is the primary event in AVM regression

(A&B) Two-photon timelapse imaging of cortical brain vessels through cranial window in Notch4* mutant mice. Vessel topology was visualized by plasma labeling by intravenous FITC-dextran. Centerline velocity in the regressing AV shunt, feeding artery (FA) and adjacent artery (AA) was obtained by direct measurement of the velocity of individual red blood cells. Repression of Notch4* decreased blood flow velocity by 48hrs in shunt and feeding artery, but increased velocity in adjacent artery. (C) Summary of % Δ in calculated flow in vessels either 48hrs after Notch4* suppression or after 48hrs with no Notch4* suppression. (D) Illustration of empirical data and hypothetical scenarios of regression. The primary event can be either the acute narrowing of the AV shunt or a reduction in flow, caused by either steal from an adjacent artery or a systemic reduction in flow. The acute AV shunt narrowing model, predicting the increase in adjacent artery flow and reduction in feeding artery flow, best fits the empirical observations. We do not observe increased feeding artery flow, as predicted by the adjacent artery “steal” model, or a decrease in adjacent artery flow, as predicted by systemic flow reduction model. Scale bars = 50μm.

Figure 3

Figure 3. Regression is initiated by the loss-less reorganization of endothelial cells

(A) Two-photon timelapse imaging through a cranial window of nuclei marked by ephrin-B2+/H2B-eGFP in a Notch4* mutant mouse. Plasma was labeled by intravenous Texas Red-dextran. In the AV shunt shown, vessel diameter was reduced by 28hrs after Notch4* repression, while the GFP+ nuclei, representing the cells, were retained at 28hrs, and even at 36hrs when vessel was further regressed. Because these images are Z-stacks through the vessel, cell 6 presented at 36hrs was also present earlier but out of the imaging plane. (B) Two-photon timelapse imaging through a cranial window of nuclei marked by Tie2-tTA/TRE-GFP in a Notch4* mutant mouse. Plasma was labeled by intravenous Texas Red-dextran. In the AV shunt shown, vessel diameter was reduced by 20hrs after Notch4* repression, while the GFP+ nuclei, representing ECs, were retained at 20hrs, and even at 28hrs when vessel was further regressed. At 36hrs further regression was evident, when some loss of was detected in the large shunt, V1, but not the smaller shunt, V2. Scale bars = 50μm.

Figure 4

Figure 4. Turning off Notch4* normalizes arteriovenous specification in AV shunts

(A–I) Whole mount LacZ staining of the surface vasculature of the cerebral cortex to reveal expression of Notch upstream venous specification gene Coup-TFII, downstream venous marker EphB4, and downstream arterial marker ephrin-B2. Perfused vessels were counterstained by colorimetric 3,3′-diaminobenzidine (DAB) reaction with horseradish peroxidase (HRP) bound tomato-lectin. (A–C) LacZ staining of Tie2-cre activated Coup-TFII reporter. (A) In control mice, Coup-TFII was expressed in the veins, venules and capillaries up to the arterioles. (B) In Notch4* expressing mutants, Coup-TFII was expressed in the vein, and venous portion of the AV shunt. (C) After repression of Notch4*, the narrowest point in AV shunts was found between Coup-TFII positive and Coup-TFII negative endothelium. (D–F) LacZ staining of EphB4 reporter. (D) In control mice, EphB4 was expressed in the veins and venules up to the capillaries. (E) In Notch4* expressing mutants, EphB4 expression was reduced through AV shunts, venules and veins. (F) Following the repression of Notch4*, EphB4 expression was increased in the regressing AV shunt. (G) In control mice, ephrin-B2 expression was detected in the arteries and arterioles up to the capillaries. (H) In Notch4* expressing mutants, ephrin-B2 expression was detected in arteries, the AV shunts, and at lower levels extending into the veins. (I) After repression of Notch4*, ephrin-B2 expression was decreased in the regressing AV shunts and veins. Closed arrowheads indicate venules; open arrowheads indicate arterioles. _n_=3 (A–C,G,I), _n_=4 (H), and _n_=8 (D–F) for each condition. Scale bars = 100μm. (J–U) Whole mount immuno-fluorescence staining of cerebral cortex after FITC-lectin perfusion. (J–L) Endothelial localization of Notch4-ICD was undetectable in control mice (J), but present in a focal manner consistent with nuclear localization throughout the artery and vein in Notch4*-On mice (arrowheads in K), and reduced once Notch4* was turned off (L). Arterial markers Dll4 (M–O), Jag1 (P–R) and Cx40 (S–U) were expressed in the artery and not vein in control mice (M,P,S). All of these markers were upregulated in the artery, through the AV shunt, and into the vein in Notch4*-On mice (N,Q,T). When Notch4* was turned off the expression in the AV shunt and vein was lost, but arterial expression remained (O,R,U). _n_=5 for all mutants, _n_=2 for each controls. Scale bars = 100μm.

Figure 4

Figure 4. Turning off Notch4* normalizes arteriovenous specification in AV shunts

(A–I) Whole mount LacZ staining of the surface vasculature of the cerebral cortex to reveal expression of Notch upstream venous specification gene Coup-TFII, downstream venous marker EphB4, and downstream arterial marker ephrin-B2. Perfused vessels were counterstained by colorimetric 3,3′-diaminobenzidine (DAB) reaction with horseradish peroxidase (HRP) bound tomato-lectin. (A–C) LacZ staining of Tie2-cre activated Coup-TFII reporter. (A) In control mice, Coup-TFII was expressed in the veins, venules and capillaries up to the arterioles. (B) In Notch4* expressing mutants, Coup-TFII was expressed in the vein, and venous portion of the AV shunt. (C) After repression of Notch4*, the narrowest point in AV shunts was found between Coup-TFII positive and Coup-TFII negative endothelium. (D–F) LacZ staining of EphB4 reporter. (D) In control mice, EphB4 was expressed in the veins and venules up to the capillaries. (E) In Notch4* expressing mutants, EphB4 expression was reduced through AV shunts, venules and veins. (F) Following the repression of Notch4*, EphB4 expression was increased in the regressing AV shunt. (G) In control mice, ephrin-B2 expression was detected in the arteries and arterioles up to the capillaries. (H) In Notch4* expressing mutants, ephrin-B2 expression was detected in arteries, the AV shunts, and at lower levels extending into the veins. (I) After repression of Notch4*, ephrin-B2 expression was decreased in the regressing AV shunts and veins. Closed arrowheads indicate venules; open arrowheads indicate arterioles. _n_=3 (A–C,G,I), _n_=4 (H), and _n_=8 (D–F) for each condition. Scale bars = 100μm. (J–U) Whole mount immuno-fluorescence staining of cerebral cortex after FITC-lectin perfusion. (J–L) Endothelial localization of Notch4-ICD was undetectable in control mice (J), but present in a focal manner consistent with nuclear localization throughout the artery and vein in Notch4*-On mice (arrowheads in K), and reduced once Notch4* was turned off (L). Arterial markers Dll4 (M–O), Jag1 (P–R) and Cx40 (S–U) were expressed in the artery and not vein in control mice (M,P,S). All of these markers were upregulated in the artery, through the AV shunt, and into the vein in Notch4*-On mice (N,Q,T). When Notch4* was turned off the expression in the AV shunt and vein was lost, but arterial expression remained (O,R,U). _n_=5 for all mutants, _n_=2 for each controls. Scale bars = 100μm.

Figure 5

Figure 5. Venous marker EphB4 is re-expressed in venous endothelial cells and required for AV shunt regression

(A) Sagittal sections showing veins in the cerebellum of Tie2-tTA; TRE-Notch4*; TRE-H2B-eGFP mutants before and 5 days after Notch4* repression, with littermate Tie2-tTA; TRE-H2B-eGFP control. EphB4 expression in TRE-eGFP+ cells, by immuno-fluorescence staining, was selectively reduced in Notch4* expressing mutant, and recovered upon Notch4* repression. Graph shows quantification of EphB4 fluorescence signal intensity in TRE-GFP+ cells. _n_=4 for mutants, n=3 for controls, an average of ~12 cells per vessel and >5vessels per mouse. (B) Two-photon timelapse imaging of cortical brain vessels through a cranial window in Notch4* mutant mice. Plasma was labeled by intravenous FITC-dextran. Treating Notch4* mutant mice with soluble EphB4 (sEphB4) inhibited the regression of the AV shunt examined over 48hrs of Notch4* repression. In a littermate Notch4* mutant treated with control soluble human fibronectin (sFN), the AV shunt was reduced in diameter following 48hrs of Notch4* repression. Quantification of changes in minimal AV shunt diameter over 48hrs in mice without repression of Notch4* (Notch4*-On, _n_=35 AV shunts in 11 mice), with repression of Notch4* (Notch4*-Off, _n_=22 AV shunts in 10 mice), with repression of Notch4* and sEphB4 intravenous treatment (+sEphB4, _n_=26 AV shunts in 5 mice), and with repression of Notch4* and sFN control intravenous treatment (+sFN control, _n_=13 AV shunts in 2 mice). Error bars represent s.e.m. between individual AV shunts.

Figure 6

Figure 6. Repression of Notch4* normalizes vascular perfusion, oxygenation, tissue structure

(A–C) Vascular perfusion of surface vessels of the cerebral cortex by fluorescent tomato-lectin. Following repression of Notch4*, capillary perfusion was increased. (D–F) Immunofluorescence (red) staining for hypoxyprobe (pimonidazole) adduct in coronal section of mouse cortex. Patches of staining were visible in mutant mice with neurologic defects before Notch4* suppression (D). Staining was reduced 72hrs after suppression of Notch4* (E). Control tissue shows an absence of staining (F). Quantification of staining intensity in cortical brain relative to non-specific IgG controls (*P<0.05 vs. all other groups). _n_=9 at 0hrs Notch4*-Off, _n_=8 at 24hrs Notch4*-Off. (G–J) Hematoxylin and eosin staining of sagittal paraffin sections of cerebellum. In Notch4*- mutant prior to Notch4* repression (0hrs Notch4*-Off), areas of hemorrhage (open arrowhead) and necrotic tissue (closed arrowhead) were visible (G). After 28 days of Notch4* suppression (Notch4*-Off), areas of scarring were visible (open arrow), but hemorrhage and necrotic tissue had been resolved (H). The numbers of purkinje cells were decreased in H when compared to these cells in the corresponding area in control J (solid arrows). Granular cells (open arrow in H) were found in the scarred area. Scale bar = 100μm.

Figure 7

Figure 7. Model for AVM regression: normalization of arteriovenous programming elicited by repression of Notch4* initiates AVM narrowing

(A) In control mice, Notch and ephrin-B2 are expressed in arteries and into capillaries. Coup-TFII and EphB4 are expressed in veins, and into capillaries. (B) In mutant mice, Notch4* is forcibly expressed throughout the endothelium, causing the repression of EphB4, and the expression of ephrin-B2 through AV shunts. The venous marker Coup-TFII, upstream of Notch, is retained, demarcating the original arterial venous boundary. (C) Repression of Notch4* allows EphB4 to be re-expressed in Coup-TFII+ venous segment. Normalization of Ephrin-B2/EphB4 signaling in the AV cell interface results in reorganization of endothelial cells, initiating the AV shunt narrowing and AVM regression.

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