Endothelial Notch4 signaling induces hallmarks
of brain arteriovenous malformations in mice
Patrick A. Murphy*, Michael T. Y. Lam*†, Xiaoqing Wu*‡, Tyson N. Kim*, Shant M. Vartanian*, Andrew W. Bollen§,
Timothy R. Carlson*¶, and Rong A. Wang*?
*Pacific Vascular Research Laboratory, Division of Vascular Surgery, Department of Surgery, and Department of Anatomy and§Department of Pathology,
University of California, San Francisco, CA 94143
Edited by Michael A. Gimbrone, Jr., Harvard Medical School, Boston, MA, and approved May 29, 2008 (received for review March 24, 2008)
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
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 neu-
ronal cell death within the cerebral cortex and cerebellum. Strik-
ingly, 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 microvascu-
lature, 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
angiogenesis ? cell signaling ? endothelial cell ? stroke ? cerebrovascular
rupture, leading to stroke and death. Historically, 50% of
BAVMs first present with hemorrhage (1). Development of
noninvasive imaging methods has increased BAVM detection
before hemorrhage, but the efficacy of current treatments,
primarily surgical resection, is questionable. Accompanying risk
of mortality (3%) and morbidity (9%) may outweigh the risk of
rupture itself (2). An ongoing clinical trial (ARUBA) is testing
the hypothesis that invasive treatment may not provide a signif-
icant improvement over the natural course of the disease (2).
BAVMs disrupt normal vessel hierarchy and are caused by the
replacement of capillary beds, which separate arteries from
veins, with enlarged and tangled vessels. The retention of
to the speculation that they form during early brain development
(3). Studies of a more accessible skin arteriovenous malforma-
tion (AVM) in human beings suggests that enlargement of
postcapillary venules precedes AVM formation (4), but exper-
imental evidence is required to validate such mechanisms. An
animal model that mimics the characteristics of human BAVMs,
principally arteriovenous shunting, would open avenues to un-
derstanding their cellular and molecular pathogenesis.
The primary factors regulating vessel hierarchy were once
thought to be hemodynamic forces (5). It is now known that
genetic programs contribute to the development of vascular
emerged as a critical genetic mediator in the differentiation of
arteries and veins. Originally discovered in Drosophila, the
pproximately 600,000 people worldwide harbor brain arte-
riovenous malformations (BAVMs) that can potentially
transmembrane Notch receptor is conserved throughout the
animal kingdom and is best known for its function in dichoto-
mous cell fate decisions through cell–cell communication (7).
Ligand binding to its extracellular domain results in sequential
cleavage events and release of an active intracellular domain
(ICD), which then translocates to the nucleus and initiates
of Notch-ICD is a hallmark of Notch activation.
In the vascular system, Notch receptors and ligands are
expressed in arteries but not veins (8) and are necessary and
sufficient to induce ephrin-B2, a faithful marker of arterial
identity (9–11). Notch loss-of-function mutations impair vascu-
lar development, resulting in arteriovenous shunting in both
zebrafish and mouse embryos (10, 12). Notch gain-of-function
also results in abnormal vascular remodeling in embryos, dem-
onstrating that proper spatial and temporal patterns of Notch
activity are critical for this process (13). Because BAVMs are
thought to occur during brain development (3), we hypothesized
that expression of constitutively active Notch4 would induce
BAVMs during the neonatal period, when areas of the brain, such
as the neocortex and cerebellum, expand by nearly 10-fold (14).
Expression of Constitutively Active Notch4 (int3) in the Endothelium
Results in Hemorrhage, Neurological Damage, and Death in Neonates.
To test our hypothesis that Notch4 activation in the endothelium
of the developing brain would cause BAVM, we expressed int3
specifically in the endothelium from birth in tetracycline-
regulated mice (Tie2-tTA;TRE-int3) (11). Endothelial-specific
expression was demonstrated by a TRE-LacZ reporter (11, 15)
[supporting information (SI) Fig. S1]. In addition, Notch4-ICD
was detected in the nuclei of a subset of mutant but not control
endothelial cells (ECs) (Fig. 1A and B). Because int3 is a portion
of Notch4, the anti-Notch4-ICD staining could not distinguish
int3 from endogenous Notch4. However, the staining did not
detect endogenous levels of Notch4 in the control, suggesting
that staining in the mutant represents int3 expression. The
activity specifically in ECs of mutant mice.
M.T.Y.L., X.W., T.N.K., and T.R.C. performed research; S.M.V. contributed new reagents/
analytic tools; P.A.M., M.T.Y.L., X.W., T.N.K., A.W.B., T.R.C., and R.A.W. analyzed data; and
P.A.M. and R.A.W. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
†Present address: Medical Scientist Training Program, University of California, San Diego,
‡Present address: Vaccine Basic Research, Merck and Company Inc., West Point, PA 19486.
¶Present address: Abbott Vascular, Abbott Laboratories, Abbott Park, IL 60064.
?To whom correspondence should be addressed at: HSW 1618, Box 0507, 513 Parnassus
Avenue, San Francisco, CA 94143-0507. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
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Mutant mice died between 2 and 5 weeks of age (Fig. 1C).
Signs of neurological dysfunction, including ataxia and seizure,
were evident in 27 of 107 mutant mice. Ataxia ranged in severity,
from mice unable to right themselves to those with splayed rear
legs. We occasionally noticed seizure episodes in which the mice
ran wildly before collapsing. All but one of the cases of ataxia
(23/24) and seizure (5/6) occurred before P22, suggesting that
younger mice are more susceptible to these phenotypes.
To investigate potential causes for the neurological dysfunc-
tion, we examined mutant brains and found prominent hemor-
rhage by gross inspection (Fig. 2B) and by histology (Fig. 2 C–F).
Hemorrhage occurred most often in the cerebellum, followed by
the neocortex, but never in brainstem. We examined six of the
ataxic mice and found hemorrhage in the cerebellum (see Fig. 2
C and D). Hemorrhage appeared more widespread than the
neurological phenotype, because we also observed hemorrhage
in all ill mutant mice without detectable ataxia (13 of 13; see Fig.
2E) and none in their littermate controls. Thrombosis, often a
consequence of hemorrhage (16), was seen in hemorrhagic areas
(see Fig. 2 D and F). As with the neurological phenotype,
hemorrhage was most severe in mice ill before P22. Blood was
found in the ventricle of 4 of 11 ill mice before P21 (see Fig. 2F).
Neuronal cell death consistently occurred adjacent to hemor-
rhage (see Fig. 2E), suggesting that hemorrhage led to neuronal
damage. Foci of pyknotic and karyorectic nuclei, often associ-
ated with macrophage infiltrations, were detected in the Pur-
kinje and granular layers of the cerebellum, as well as areas of
the neocortex and midbrain (see Fig. 2E). Signs of calcification,
an indication of earlier cerebral damage, occurred in multiple
cases (data not shown). Therefore, increased endothelial Notch4
activation results in hemorrhage, which likely causes neuronal
damage and ataxia in the mutant mice.
Vessel Enlargement and Hallmarks of BAVMs in Mutants Before
Hemorrhage. Because increased Notch4 activation was targeted
to ECs specifically, we investigated lesions underlying hemor-
rhage and found vascular abnormalities resembling BAVMs.
Vascular perfusion with casting agents (Fig. 3B) or fluorescent
lectins (Fig. 3D and Fig. S2) revealed enlarged and tangled
vessels, hallmarks of BAVMs (1, 17). We verified that arteries,
arterial and venous branches of the middle-cerebral vessels (see
Fig. S2). To determine whether the enlarged vessels had smooth
muscle coverage, we performed immunofluorescence with anti-
smooth muscle ?-actin (SMA) and found strong staining in these
vessels (Fig. S3). Vessel enlargement occurred before hemor-
rhage, neuronal cell death, or signs of neurological dysfunction
(Fig. S4), suggesting that it is a primary defect.
Because hemorrhage occurred primarily in the cerebellum,
followed by the neocortex, and never in brainstem, we tested
whether vessel enlargement correlated with hemorrhage. A
decrease in the proportion of small vessels and an increase in the
proportion of larger vessels were most significant in the cere-
bellum, followed by the neocortex, and least in brainstem (Fig.
4). Therefore, regional enlargement correlated with hemor-
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 ar-
rowheads). (C) Kaplan–Meier survival curve shows that all mutants died by
P36. (Scale bar, 50 ?m.)
Endothelial expression of int3 causes ataxia and death in neonatal
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 archi-
tecture (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 ?m; D, 100 ?m; E, 200 ?m.)
Brain hemorrhage occurred in all mutant mice. (A and B) Multifocal
and midbrain by vascular casting at P27 (B; white arrowheads) and in the
(A and C) Abnormal vessels were not observed in littermate controls. (Scale
bar, 200 ?m.)
Enlarged and tangled blood vessels developed in all mutant mice.
www.pnas.org?cgi?doi?10.1073?pnas.0802743105Murphy et al.
rhage, supporting the hypothesis that vessel enlargement con-
tributes to hemorrhage.
Endothelial Notch activity decreases vessel density by inhib-
iting sprouting angiogenesis (18). To investigate vessel density,
we quantified CD31 positive vessels. Vessel density was de-
creased significantly in the cerebellum, and progressively less so
in the neocortex and brainstem (see Fig. 4). Vessel density was
inversely correlated with the proportion of large vessels (?50
?m) across all of these regions (Fig. S5), suggesting that vessel
enlargement is linked to the reduction in vessel density.
Arteriovenous Shunting Occurs in all Mutants. The fundamental
defect in human BAVMs is high-flow arteriovenous shunting.
Blood velocity in carotid arteries can double in BAVM patients,
a clinical indication of arteriovenous shunting (19). We mea-
sured carotid blood velocity with high-resolution ultrasound and
found increased carotid blood velocity in all mutants by 3 weeks
of age (Fig. 5A). This noninvasive approach allowed for pro-
gressive measurements in the same animal, demonstrating that
carotid blood velocity increased in a short time window between
P17 and P21 (see Fig. 5A). Furthermore, at P21, velocity in
mutants with neurological dysfunction was significantly higher
than those without neurological dysfunction (2.6 ? 0.5 m/sec;
n ? 6 versus 1.9 ? 0.5 n ? 20, respectively; P ? 0.007). Because
of limitations of this assay, we were unable to examine sick
mutants before this age, and they often exhibited severe neu-
rological defects. As a result, the cohort of mice examined did
not display severe neurological defects, which may contribute to
the minimal increase at P15.
To more directly test for arteriovenous shunts, we performed
a microsphere passage assay (11). Microspheres lodged within
the control brain (Fig. 5B) but bypassed the mutant brain and
lodged in the downstream lung (Tie2-tTA n ? 6; Tie2-tTA;TRE-
int3 n ? 5). This result demonstrates the existence of arterio-
venous shunts in the brain, mimicking the principal defect of
To directly visualize arteriovenous shunts, we distinguished
arterial from venous branches with the arterial marker ephrin-
B2. Expression of the reporter in ephrin-B2?/tLacZmice (6) was
strong in the middle cerebral arteries and weak in the middle
cerebral veins (Fig. 6A). In mutants, direct connections between
the arteries and veins were prominent at P21 (Fig. 6B). Similar
arteriovenous connections within the cerebellum were detected
by using a nuclear GFP reporter in ephrin-B2?/GFPmice (20).
Cerebellar interfolial arteries (GFP?) in the control follow a
typical pattern of branching and ramifying into a fine capillary
bed within the granular layer before coalescing into the draining
veins (GFP?) (Fig. 6C) (21). However, in mutants there were
enlarged vascular connections, replacing normal capillaries,
between the interfolial arteries and veins (Fig. 6D). Therefore,
direct arteriovenous shunts were visible in the surface and deep
cerebellar vasculature of mutant mice.
Repression of int3 Rescues Moribund Mice. Taking advantage of the
tetracycline-regulated expression system, we tested whether
frequency of hemorrhage. Shown is immunofluorescence in sagittal sections
CD31 immunostained (black) vessels in each region (per squared millimeter).
? Proportion of vessel with given diameter represents the percent change in
the proportion of small (?7.5 ?m), medium (7.5–20 ?m), and large (?20 ?m)
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
(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 ?
P21 (***, P ? 0.00005). Typical Doppler traces from mice at P21 (A Right). (B)
Brain arteriovenous shunts developed by P19 as shown by fluorescent micro-
sphere passage. The microspheres bypassed the brain and lodged in the lung
in the mutants but not controls. BF, Bright-field; FITC, green fluorescent
All mutants developed shunting and arteriovenous malformations.
Murphy et al.
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repression of the causal genetic lesion could reverse disease
progression in mutant mice. Vascular enlargement and shunting
occurred in all mutant animals by P21, so this time-point was
chosen for a regression study. Littermates were allowed to
express the int3 transgene from birth until P20 or P21, at which
time sick mutants were fed doxycycline (Dox), a more stable
derivative of tetracycline (Tet), to repress int3 expression. No
mutants survived to P32 without Dox treatment (n ? 19), but 8
of 9 mutants fed Dox appeared outwardly healthy and active at
P35, and were healthy until killed by P64 (Fig. 7A). The one
exception was a severely affected mutant that died in the first day
To investigate the reversal of brain abnormalities specifically,
examined was resolved within just days of transgene repression
(Fig. 7B). These findings suggest that repression of endothelial
int3 expression, the causal stimulus, recovers brain function in
We demonstrate that increased Notch4 activity causes vascular
abnormalities with the characteristics of BAVM, implying that
increased Notch4 activation is a potential molecular cause of
human BAVM. Our findings also suggest that regression of this
devastating disease may be possible by targeting the causal
molecular lesion. The activated Notch4 model provides a pow-
erful platform for the study of the pathogenesis and regression
of BAVM-like lesions. In addition, our studies of pathogenesis
in this model provide experimental support for the hypothesis
that BAVMs occur in the developing brain and that they can be
induced by vessel enlargement.
Notch4 Activation Is a Candidate in the Molecular Regulation of BAVM
Pathogenesis. Molecular etiology of BAVM is unknown. Muta-
tions in the TGF-? signaling pathway, primarily loss-of-function
ENG (endoglin) and ACVRL1 (ALK-1) alleles, have been asso-
ciated with BAVMs (22). However, these mutations are impli-
cated in only ?2% of all BAVMs (23). We found that consti-
tutively active Notch4 causes BAVM-like lesions promptly in
neonates, identifying activation of the Notch4 pathway as a
potent molecular candidate in the development of human
The primary characteristic of human BAVMs is the develop-
ment of arteriovenous shunting (1). In patients, this is detected
by arteriography, the gold standard in the diagnosis of this
vascular lesion (24), and also by ultrasound measurement of
increased carotid blood flow (25). We demonstrated the pres-
ence of arteriovenous shunting by similar methods in mice,
including anatomical connections between arteries and veins,
functional passage of beads too large for capillaries, and in-
creased carotid blood velocity.
Enlarged and tangled vessels are another key characteristic of
BAVMs (1). We detected massively enlarged and tangled vessels
by perfusion labeling with a casting agent and fluorescently
labeled lectins. In contrast to human BAVMs, which usually
develop as a single nidus (23), the vascular abnormalities in
because of the expression of the int3 transgene throughout the
Hemorrhage, often resulting in neuronal damage and ataxia,
is the most feared consequence of human BAVM (26). All of the
sick mutants examined developed brain hemorrhage, often with
adjacent neuronal cell death. Approximately 25% of the mice
developed neurological deficits, including ataxia and seizure.
Repression of int3 Expression Leads to Recovery of Sick Mice. It is
commonly thought that natural regression of BAVMs does not
occur (27); instead they increase in size and eventually rupture
(26). Regression of BAVMs has been reported in only a handful
of cases, which are thought to be caused by thrombotic occlusion,
a naturally occurring event similar to the embolizing treatments
used clinically (27). The recovery of brain function in our mutant
mice that resulted from molecular intervention is provocative.
One potential caveat is that Dox, which is used to repress
transgene expression, can also inhibit both vessel growth and
hemorrhage (28–30), and may therefore contribute to recovery.
Because the dose of Dox for transgene repression is similar to
surface and in the parenchyma of mutant brains. (A and B) X-gal staining of
ephrin-B2?/tLacZbrains revealed the middle cerebral artery (MCA, red arrow-
arterial branches of the MCA (A) and venous vessels (V) in Tie2-tTA;TRE-
int3;ephrin-B2?/tLacZmice (B?). (C and D) Fluorescent images of sagittal cere-
bellar 100-?m thick sections show enlarged connections (white arrowheads)
between GFP-labeled interfolial arteries of Tie2-tTA;TRE-int3;ephrin-B2?/tLacZ
Enlarged arteriovenous connections developed on the meningial
sion 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.
Repression of int3 resolved ataxia and prevented death. (A) Repres-
www.pnas.org?cgi?doi?10.1073?pnas.0802743105 Murphy et al.
that used for inhibition of vessel growth, we are currently unable
to delineate these two effects. However, similar Tet-regulated
mouse genetic approaches were used to show that tumors regress
after repression of transgenic oncogene expression, inspiring the
oncogene addiction theory (31–33). It is possible that Notch4-
induced BAVM-like lesions require continuous Notch4 activa-
tion for their maintenance.
repression of transgene expression or because of its direct effect
on vessel growth and hemorrhage, the recovery of the ill mice is
evocative. Although further studies are required to determine
whether this is a result of regression of the BAVM-like lesions,
lesion may lead to regression of the disease.
High Penetrance and Rapid Onset of BAVM-Like Lesions in the Acti-
vated Notch4 Mutants Provide a Robust Platform to Model Human
BAVM Pathogenesis. Our transgenic mice provide a much needed
mouse model for the study of BAVM pathogenesis. The study of
the cellular and molecular etiology of BAVMs is limited by the
lack of a good animal model for this disease (34). Mice with
mutations in endoglin or ALK-1 have advanced the understand-
ing of molecular contributions to AVMs. Autosomal dominant
mutations in these genes are responsible for the human hered-
itary hemorrhagic telengectasia (HHT) (22), which involves
AVMs primarily in the skin, liver and lung, along with a 4%
incidence in the brain (35). Of 22 endoglin?/?mice that devel-
oped an HHT phenotype, 8 showed signs of brain hemorrhage
by ?40 weeks of age (36). Vascular brain casts showed small
clusters of capillaries or aneurismal dilations in 3 of 10 endog-
lin?/?mice (37). Cerebrovascular abnormalities are also ob-
served in ?2% of Alk-1?/?mice (38). However, it is unknown
whether either the endoglin?/?or Alk-1?/?mice develop arte-
riovenous shunting. Therefore, our activated Notch4 mice pro-
vide a model for molecular understanding of both the develop-
ment and potentially the regression of BAVMs.
Notch4 Activity May Selectively Induce BAVM-Like Lesions in Growing
Brains. BAVMs typically affect children and younger people.
Based primarily on the belief that BAVMs retain embryonic
vascular structures, it has been speculated that BAVMs occur
during brain growth (26). Our data supports the theory that the
growing brain is more vulnerable to BAVM formation. First, we
found that hallmarks of BAVM are prominent when the trans-
gene is expressed in the neonatal period, when the brain grows
rapidly, but not when it is expressed in the postweaning mouse
(11), when brain growth is reduced. Second, we found that vessel
enlargement was greatest in the cerebellum and cortex and least
in the brainstem, which correlates with the greater growth in the
cortex and cerebellum than in the brainstem at this stage (39).
Third, when AVMs occur in older mice, they appear to affect the
liver, skin, and uterus, three actively remodeling tissues in adult
mice (11). These data support the hypothesis that Notch func-
tions in a temporal and spatial specific manner, and that the
developing brain is particularly susceptible to Notch4-mediated
formation of BAVM-like lesions.
cellular mechanism of BAVM formation is currently unknown.
A few models, however, are proposed. The most favored model
is that BAVMs represent a failed transition from embryonic
brain vasculature to adult brain vasculature (3). Supporting
evidence for this theory has rested heavily on the interpretation
of the morphological structure of adult BAVMs. Another theory
is that BAVMs are not necessarily congenital abnormalities, but
can form de novo through vessel enlargement. This theory is
based on the observation that enlargement of postcapillary
venules is the first observable phenotype in the development of
AVMs in human skin (4).
Our data support the theory of de novo formation of BAVMs
through vessel enlargement in the context of growing brain. We
are able to measure carotid blood flow, a sensitive indicator of
brain arteriovenous shunting, longitudinally in our mutant mice.
By this measurement, we were able to show that in individual
mutant mice, there was no shunting at P15, but that shunting
increased dramatically over the subsequent 6-day period. In
addition, the diameters of all vessels, including those at the
capillary level, were enlarged. The enlargement of vessels before
brain hemorrhage or neurological phenotypes suggests that it
plays a causal role in the development of BAVM-like lesions.
Thus, our data support the hypothesis that vessel enlargement
promotes the development of BAVMs.
An alternative possibility is that Notch activation disrupts the
arteriovenous interface by increasing arterial specification. We
and others have shown that Notch activity promotes arterial
specification in ECs in vivo in embryonic and adult mice, as
determined by the expression of arterial markers, such as
ephrin-B2 (9–11). In this study we found that in neonatal brain
endothelium, Notch4 activity increased ephrin-B2 expression in
capillaries and veins. Interestingly, expression was most mark-
edly increased in direct arteriovenous connections, suggesting
that induction of arterial specification in these ECs may disrupt
the proper formation of the capillary bed connecting the artery
Notch Activity May Promote Vessel Enlargement by Inhibiting Sprout-
ing. We previously reported that Notch activity promotes vessel
enlargement (11), and others subsequently demonstrated a
reduction in vessel density (18), but the relationship of these
functions is unclear. We observed a correlation between vessel
enlargement and vessel density, suggesting that Notch’s role in
enlargement and BAVM development. Supporting our finding,
it was recently reported that retroviral expression of the Notch
ligand Dll4 increases lumen diameters and reduces vessel density
in grafted tumors in mice (40). The observation that impaired
endothelial sprouting can result in vessel enlargement was first
made in mice exclusively expressing the VEGF120isoform (41).
In both the VEGF mutant and our previous studies (11),
proliferation was not significantly altered, suggesting that in-
creased vessel size may simply reflect the retention of cells that
would have otherwise contributed to sprouts. Therefore, Notch
activation may promote vessel enlargement by inhibiting vessel
In summary, we believe that this work opens an area of
research to advance the molecular understanding of BAVM
pathogenesis and provides hope for the treatment of this dev-
Mice. Tet sucrose solution (0.5-mg/ml Tet, 50-mg/ml sucrose, Sigma) was
administered to pregnant mothers from plugging, and withdrawn from pups
at birth, and Dox (200-mg/kg diet, Bio-Serv) diet was administered to mutant
mice at P20–21 as we described (11, 42). All animals were treated in accor-
dance with the guidelines of the University of California San Francisco Insti-
tutional Animal Care and Use Committee.
as we described (11). Casting and FITC-lectin staining were performed as we
described (11). Cy3-streptavidin (Jackson Immuno) was used to detect biotin-
?g of each in 200-?l PBS). Immunostaining was performed with anti-CD31,
anti-?-SMA, and anti-Notch4 (Upstate) according to our published protocols
Murphy et al.
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