Linking Notch signaling to ischemic stroke
Joseph F. Arboleda-Velasquez*, Zhipeng Zhou†, Hwa Kyoung Shin†, Angeliki Louvi‡, Hyung-Hwan Kim§, Sean I. Savitz†,
James K. Liao§, Salvatore Salomone†, Cenk Ayata†, Michael A. Moskowitz†, and Spyros Artavanis-Tsakonas*¶?
*Department of Cell Biology, Harvard Medical School, Boston, MA 02115;†Stroke and Neurovascular Regulation Laboratory, Massachusetts General
Hospital, Boston, MA 02129;‡Program on Neurogenetics and Department of Neurosurgery, Yale University School of Medicine, New Haven, CT 06520;
§Vascular Medicine Research Unit, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, MA 02139; and¶Colle `ge de France,
75231 Paris, France
Edited by Pietro V. De Camilli, Yale University School of Medicine, New Haven, CT, and approved January 15, 2008 (received for review October 16, 2007)
Vascular smooth muscle cells (SMCs) have been implicated in the
pathophysiology of stroke, the third most common cause of death
and the leading cause of long-term neurological disability in the
world. However, there is little insight into the underlying cellular
pathways that link SMC function to brain ischemia susceptibility.
Using a hitherto uncharacterized knockout mouse model of Notch
3, a Notch signaling receptor paralogue highly expressed in vas-
cular SMCs, we uncover a striking susceptibility to ischemic stroke
upon challenge. Cellular and molecular analyses of vascular SMCs
derived from these animals associate Notch 3 activity to the
expression of specific gene targets, whereas genetic rescue exper-
iments unambiguously link Notch 3 function in vessels to the
ischemia ? Notch3 ? vascular smooth muscle ? CADASIL
(1, 2). The central element of this signaling pathway is the Notch
cell surface receptor, a single-pass transmembrane protein,
which interacts with membrane-bound ligands expressed on
adjacent cells linking the fate of one cell to that of its neighbor
(3, 4). In mammals, four paralogs of the Notch receptor have
been identified, Notch 1–4, with overlapping but nonidentical
expression patterns (5). In adult tissues, Notch 3 expression is
restricted to vascular smooth muscle cells (SMCs) (5). Notwith-
standing subtle arterial abnormalities reported in Notch 3 mu-
tant mice (6), the role of Notch 3 in vascular physiology remains
otch signaling defines one of the fundamental cell interac-
tion mechanisms governing cell fate choices in metazoans
To explore Notch 3 function, we used a previously uncharacter-
ized Notch 3 knockout mouse model provided by W. C. Skarnes
and M. Tessier-Lavigne (7). Like Notch 3 knockouts studied in
refs. 8 and 9, this mutant mouse was viable and fertile. The null
allele was generated by insertional mutagenesis with a lacZ
carrying vector so that ?-galactosidase (?-gal) expression par-
allels that of Notch 3 (Fig. 1). Our analysis generally agrees with
reported Notch 3 expression studies in the vasculature (10) but
did reveal a broader distribution, including the neuronal pro-
genitor-containing ventricular zone of the developing neural
tube between embryonic day 12.5 (E12.5) and E15.5 and the
neonatal brain [supporting information (SI) Appendix, SI Fig. 6,
and data not shown]. Relevant to this study, in situ hybridization,
X-gal staining, and immunofluorescence confirmed expression
of Notch 3 in SMCs from brain vessels and aorta (Fig. 1 D–F and
data not shown). A morphological study involving immunostain-
ing with SMC-specific antibodies and electron microscopy did
not reveal any abnormalities in either brain vessels or the aorta
of knockout mice (Fig. 1G; SI Appendix, SI Fig. 7; and data not
To investigate further the properties of SMCs lacking Notch
3 function, we developed a FACS-based cell purification proto-
col, taking advantage of the ?-gal expression associated with the
Notch 3 knockout allele in SMCs, virtually the only cells in the
adult brain to express Notch 3 (Figs. 1 and 2). Brain-derived cells
from Notch 3?/?or Notch 3?/?mice were isolated and shown to
2 B and C and SI Appendix, SI Fig. 8). Availability of a highly
enriched population of SMCs allowed us to examine the impact
of Notch 3 loss-of-function on the transcriptional profile of
brain-derived SMCs (BrSMCs). Comparative analysis between
Notch 3?/?and Notch 3?/?cells revealed 662 differentially
regulated genes, using an arbitrary cutoff (P ? 0.01, fold
change ? 1.5). Notch 3 scores as the utmost down-regulated gene
(?22.1-fold) in Notch 3?/?BrSMCs. Indicative of their abnor-
mal Notch signaling capacity, the canonical Notch downstream
targets Heyl and Hes1 were down-regulated (both ?1.5-fold).
Gene ontology analysis of all misregulated targets in BrSMCs
showed statistical overrepresentation of genes classified under
four functional categories named ‘‘muscle contraction’’ (all
down-regulated), ‘‘cell structure and motility,’’ ‘‘muscle devel-
opment,’’ and ‘‘mesoderm development’’ (SI Appendix, SI Tables
1 and 2), consistent with the notion that SMCs from knockout
animals harbor significant functional differences compared with
those carrying WT Notch 3 receptors.
Given the relevance of vascular SMCs to stroke, we examined
the ischemia susceptibility of mice lacking Notch 3 function in a
standard filament model of proximal middle cerebral artery
(MCA) occlusion (11). In this assay, Notch 3?/?mice developed
ischemic lesions approximately twice as large as those seen in
WT or heterozygous (Notch 3?/?) 10- to 12-week-old male mice
(Fig. 3 A and B). Consistent with the severity of stroke, neuro-
logical deficits were more pronounced (Bederson neurological
score on day 1 median values: WT ? 1, Notch 3?/?? 2, P ? 0.01)
and mortality higher upon MCA occlusion in Notch 3?/?mice
compared with WT (Fig. 3C). To assess whether enlarged
deficits, we used laser speckle flowmetry (LSF) during distal
MCA occlusion (12). This two-dimensional optical imaging
technique measures cortical blood flow with high spatial reso-
lution, quantifies the ischemic area, and allows for monitoring of
spontaneous periinfarct depolarizations (PIDs) triggered by
anoxic release of K?and excitatory amino acids from the infarct
core (13, 14). Using this method, we found that Notch 3?/?mice
developed a 60% larger area of severe CBF deficit than WT mice
(P ? 0.01) (Fig. 3 D and E). Thus, using two distinct approaches,
we find complete loss of Notch 3 function to be associated with
significant ischemic abnormalities. Interestingly, the frequency
of spontaneous PIDs, which are known to aggravate stroke (15),
S.S., C.A., M.A.M., and S.A.-T. designed research; J.F.A.-V., Z.Z., H.K.S., A.L., H.-H.K., S.I.S.,
S.S., and C.A. performed research; J.F.A.-V., Z.Z., H.K.S., A.L., H.-H.K., S.I.S., J.K.L., S.S., C.A.,
M.A.M., and S.A.-T. analyzed data; and J.F.A.-V., A.L., C.A., M.A.M., and S.A.-T. wrote the
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
?To whom correspondence should be addressed. E-mail: email@example.com.
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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was more than doubled in Notch 3?/?compared with WT mice
(6.0 ? 2.5 vs. 2.9 ? 2.5 PIDs per h, P ? 0.05) (Fig. 4). However,
Notch 3?/?mice did not exhibit the characteristic transient
hypoperfusion episodes during PIDs, suggesting that the vaso-
constrictive ability of cerebral vessels was impaired in the
mutants, an observation congruent with the down-regulation of
muscle contraction genes unveiled by microarray analysis (Fig.
4). Systemic physiological variables (SI Appendix, SI Table 3) and
absolute resting CBF (134 ? 45 ml?100 g?1?min?1in Notch 3?/?,
136 ? 29 ml?100 g?1?min?1in WT) and circle of Willis anatomy,
examined by carbon black perfusion for the presence of com-
municating arteries (SI Appendix, SI Fig. 9), did not reveal any
differences between WT and Notch 3?/?mice that would explain
the ischemic susceptibility.
To link unambiguously the ischemic phenotype with Notch 3
function, we deemed it essential to examine whether expression
phenotype. To that end, we generated a conditional transgenic
mouse, ROSA NOTCH 3, which, when crossed to an appropriate
Cre line [SM22-Cre (16)] could sustain SMC-specific expression
of a human NOTCH 3 transgene (Fig. 5). We found that
expression of WT NOTCH 3 in vascular SMCs of knockout mice
reduced infarct volume after filament occlusion of MCA (Fig. 5
E and F). Thus, Notch 3 expression in SMCs is both necessary
and sufficient to rescue stroke susceptibility in knockout mice,
directly linking the ischemic phenotype with Notch 3 function in
neurological disability. Although not completely understood,
extensive studies indicate that stroke burden varies greatly
depending on complex interactions between blood vessels and
brain cells (17). Here, we clearly link Notch signaling to ischemic
stroke and raise the possibility that Notch 3 defines a key
determinant of stroke burden through regulation of vascular
Extensive functional studies of contractile activity in isolated
aortas did not reveal differences between Notch 3 knockout and
WT animals (SI Appendix, SI Fig. 10). In contrast, abnormalities
in contractile tone in cerebral vessels are suggested by the lack
of vasoconstrictive response to PIDs during ischemia in Notch
3?/?animals. Consistent with these data, the microarray analysis
links Notch 3 expression in SMCs from cerebral arteries with
genes involved in vascular tone (SI Appendix, SI Tables 1 and 2),
whereas no such link could be established when SMCs from
aortas were used (data not shown). Whether the differences
between brain and aorta SMCs revealed by these studies reflect
genuine molecular phenotypic characteristics and distinct
physiological properties remains to be determined.
The relevance of this study to human stroke is exemplified by
the fact that NOTCH 3 mutations, of obscure functional nature,
are the only known cause of cerebral autosomal dominant
arteriopathy with subcortical infarcts and leukoencephalopathy
(CADASIL), a paradigmatic neurological disease characterized
by vascular SMC pathology, progressive brain ischemia, and
(Lower) containing EGF-like repeats 1–21 of Notch 3 fused to ?-gal (blue box). (B) Long-range PCR amplified intron 16–17 (?2 kb) of the Notch 3 gene in DNA
samples from WT (Notch 3?/?) or heterozygous animals (Notch 3?/?) but failed to amplify the larger intron containing the trapped vector in DNA from knockout
mice (Notch 3?/?). (C) The Notch 3 intracellular domain was detected by Western blot analysis of cultured aortic smooth muscle cells (SMCs) derived from WT
and Notch 3?/?but absent (also by qRT-PCR, data not shown) in those derived from Notch 3?/?mice. (D) Notch 3 expression. In situ hybridization, using a Notch
3 antisense riboprobe labeled vessels in brain from WT mice (Upper). Likewise, X-gal staining (Lower) of Notch 3?/?(Left) and Notch 3?/?(Right) brain vessels.
(E) Immunofluorescence of brain tissue sections demonstrated the colocalization of ?-gal and Notch 3 extracellular epitopes in brain arteries from Notch 3?/?
mice. (F) Aortic SMC layers from WT mice (white) showed Notch 3 expression. (G) Low magnification electron micrographs of arterial cortical vessels (Left) and
aorta (Right) from 8-week-old WT and Notch 3?/?mice. Asterisks indicate smooth muscle cells. L, lumen. (Scale bars: Left, 5 ?m; Right, 10 ?m.)
Characterization of the Notch 3 knockout mice. (A) A schematic of the heterodimeric Notch 3 receptor (Upper) indicating key structural features. In the
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vascular cognitive impairment (18, 19). Although vascular ab-
normalities in CADASIL are widespread, anatomical studies
have shown a predilection of NOTCH 3-associated pathology for
small vessels particularly in those brain regions with lowest blood
flow values, such as the white matter (20).
The availability of appropriate mouse models to study the role
is a prevalent cause of stroke and vascular cognitive impairment
in humans and because such models may prove valuable in
increasing our general understanding of the cellular and molec-
ular mechanisms underlying stroke pathophysiology.
Materials and Methods
Animal Protocols. Animal care and experimental procedures were performed
with approval from institutional animal care and use committees of Massa-
chusetts General Hospital, Harvard Medical School and Yale University.
Littermates were used for comparative analysis throughout.
In Situ Hybridization and Detection of ?-Galactosidase Activity. Animals were
removed, fixed overnight in 30% sucrose and 4% paraformaldehyde, and
sectioned in the coronal plane on a sledge cryomicrotome (Leica SM2000) at
40 ?m. In situ hybridization was essentially performed as described in refs. 21
and 22. X-gal staining was performed overnight at 30°C in a solution contain-
ing 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 20 mM
MgCl2, 0.1% Triton X-100, and 0.37 mg/ml X-gal.
Purification of Brain-Derived SMCs. Ten- to 12-week-old male mice were killed
and perfused with 10 ml of PBS before brain dissection. After removal of the
meninges, brain tissues were fragmented with a razor blade and digested in
10 ml of PBS (without calcium or magnesium) supplemented with a collage-
nase/dispase mixture (100 ?g/ml, Roche), incubated for 75 min at 37°C, and
homogenized by using a 10-ml pipette. Undigested material was removed by
using a 100-?m cell strainer. The flow-through was centrifuged at 4°C for 3
min at 834 ? g and the pellet washed with 10 ml of PBS (with calcium and
red; Invitrogen) before staining with fluorescein di-?-D-galactopyranoside
(FDG), a fluorogenic substrate for ?-gal (Fluka), using standard methodology
(23) modified as follows: FDG stock was prepared in a H2O:Ethyl:DMSO (8:1:1)
solution to a final concentration of 30 mM and kept frozen at ?20°C. For
staining, 2 mM FDG (in 100 ?l of H2O) and cells (in 100 ?l of medium; 1 ? 107
cells per ml) were preincubated for 5 min at 37°C, mixed together, and then
incubated for 1 min at 37°C to induce FDG uptake. The reaction was stopped
by adding 0.8 ml of Opti-MEM and incubation on ice. Individual cell prepa-
?-gal positive cells, FDG is metabolized to fluorescein allowing FACS sorting
for 1–7 days in culture (F12/MEM and 10% FBS; Invitrogen) or fixed with 4%
paraformaldehyde before immunofluorescence studies. Purity of the SMC
preparations was determined by using X-gal staining, and antibodies specific
for Notch 3 and ?-smooth muscle actin (see SI Appendix), thus they may also
Microarray Studies. Biotinylated cRNA samples from freshly sorted brain SMCs
(four Notch 3?/?mice and five Notch 3?/?mice) were fragmented before
hybridization (15 ?g each) onto mouse 430 2.0 Affymetrix chips. The chips
from Notch 3?/?(?-gal positive) but not from WT mice (?-gal negative) upon incubation with the fluorogenic ?-gal substrate fluorescein di-?-D-
galactopyranoside (FDG) (Center). Most fluorescein-positive cells (96.1%) were viable as demonstrated by propidium iodide (PI) exclusion but were heteroge-
neously distributed in the FSC-A vs. SSC-A profile (Right and data not shown). In 20 independent FACS analyses performed by using our brain digestion and FDG
staining protocols (including Notch 3?/?and Notch 3?/?samples), the percentage of PI-positive events in the total population ranged from 0.3 to 1.5%.
Fluorescein signal was only occasionally higher in brain cell suspensions derived from Notch 3?/?mice (two copies of ?-gal) compared with that of Notch 3?/?
(one copy) (Lower) consistent with a documented nonlinear relationship between fluorescence intensity and intracellular ?-galactosidase activity. (B) Seventy
to 80% of fluorescein-positive sorted cells in culture showed X-gal staining. (C) ?-gal positive cells always expressed smooth muscle-specific alpha actin epitopes
Isolation of vascular smooth muscle cells from brain. (A) FACS analysis detected significant fluorescein-positive events in brain-derived cell suspensions
www.pnas.org?cgi?doi?10.1073?pnas.0709867105Arboleda-Velasquez et al.
day as described in ref. 24. For data normalization, all probe sets were scaled
to a target intensity of 150. Microarray data analysis was performed by using
Rosetta Resolver. All cells were from 10- to 12-week-old male mice.
Gene Ontology Analyses. PANTHER software was used to define over- and
analysis (25). P values were calculated by using binomial statistics.
Model of Focal Cerebral Ischemia. Animals were anesthetized with 2% isoflu-
rane and maintained on 1.5% isoflurane in 70% N2O and 30% O2by a face
mask. Cerebral infarcts were produced by 1 h of MCA occlusion followed by
reperfusion as described in refs. 11, 26, and 27. Regional CBF and physiologic
parameters were monitored as described in refs. 11, 26, and 27. Infarct
volumes were calculated by integrating the infarct area in each brain section
of the brain, using the indirect method to correct for edema.
Determination of Infarct Size. After kill, cerebral infarct sizes were determined
by means of an image analysis system (M4; Imaging Research) as described in
refs. 11, 26, and 27.
Neurological Evaluation. Mice that underwent 1 h of fMCAO were evaluated
for neurological deficits over a period of 1 week. Deficits were measured on
a well established five-point neurological scale (28): 0, no neurologic deficit;
1, failure to extend the left forepaw fully; 2, circling to the left; 3, falling or
leaning over to the left; 4, no spontaneous walking and a depressed level of
consciousness; or 5, dead. All animals tested had a score of 0 before under-
Laser Speckle Flowmetry (LSF). Adult mice were anesthetized with isoflurane
(2% induction, 1% maintenance), endothracheally intubated, and ventilated.
Blood pressure and heart rate were continuously recorded by using PowerLab
was regularly checked by the absence of a blood pressure response to tail pinch.
skull surface was prepared for LSF to study the spatiotemporal characteristics of
3?/?(N3?/?), and Notch 3?/?(N3?/?) mice analyzed 22 h after a 1-h transient filament middle cerebral artery occlusion (fMCAO). Both infarct area and volume
were substantially larger in Notch 3?/?mice compared with those of WT and Notch 3?/?mice (10- to 12-week-old male mice, n ? 9 per group; P ? 0.01). (C) A
over 7 days, compared with no mortality in WT mice. (D) Representative laser speckle contrast images taken 1 h after distal MCA occlusion (dMCAO) are shown
from WT and Notch 3?/?mice. Distal MCA was clipped through a small temporal craniotomy (arrows). Superimposed areas (blue) indicate regions with severe
cerebral blood flow (CBF) deficit (i.e., ?20% residual CBF). Notch 3?/?mice developed significantly larger area of severe CBF deficit compared with WT. The
CBF ?20%), moderate (21–30%), and mild (31–40%) CBF deficit in WT and Notch 3?/?mice 60 min after dMCAO. The area of severe CBF deficit was significantly
larger in Notch 3?/?animals compared with WT (P ? 0.01), whereas the areas of moderate or mild CBF deficit did not differ between the two genotypes (P ?
0.05; two way ANOVA for repeated measures). Error bars indicate standard deviations.
Stroke susceptibility of Notch 3 knockout mice. (A and B) Infarct volume (indirect method) and infarct areas in individual coronal slices in WT, Notch
dMCAO (at time 0) in severe (black), moderate (green), or mildly ischemic cortex (red) in WT and Notch 3?/?(N3?/?) mice. Black dots indicate spontaneous
during the PIDs (arrows) was absent in Notch 3?/?mice.
Abnormal CBF changes upon ischemic challenge in Notch 3 knockout mice. Representative tracings showing the time-course of CBF changes after
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cerebral blood flow (CBF) changes during focal ischemia. Focal ischemia was
induced by clipping the MCA and LSF imaging was initiated 1 min before MCA
ligation and continued up to 90 min. Images obtained by a CCD camera posi-
tioned above the head were analyzed by using three separate paradigms to
ischemic cortex, and the CBF profile between the nonischemic cortex and the
ischemic core (12).
to determine tissue-specific requirements for Notch 3 expression. For this
Lasserve(InstitutNationaldelaSante ´,etdelaRechercheMe ´dicaleU740,Paris,
France) was subcloned into a vector designed for site-specific recombination
into the ubiquitously expressed ROSA26 mouse locus (29). In the final con-
struct, NOTCH 3 was flanked by a loxed stop cassette at the 5?end (for
Cre-mediated regulation of expression) and an IRES-nuclearGFP sequence at
the 3? end. The resulting construct was sequenced and electroporated into ES
cells (129SV/J line) before selection of positive clones by long-range PCR and
Southern blot hybridization. Chimeras generated through embryo injections
of ES cells clones were crossed to C57BL/6 mice to obtain germ-line transmis-
sion. The resulting ROSA NOTCH 3 knockin mice (WT76 line) were viable and
fertile and displayed no gross abnormalities.
ACKNOWLEDGMENTS. We thank Michael Waring and the Harvard Medical
School Center for AIDS Research Immunology Core at Massachusetts General
Hospital for help with cell sorting and optimization of the FDG-based purifi-
cation methods; Charles Vanderburg, Rachel Diamond, and the Harvard Cen-
ter for Neurodegeneration and Repair’s Advanced Tissue Resource Center for
RNA preparation and analysis; Christoph Rahner and the Center for Cell and
Molecular Imaging at Yale University School of Medicine for help with elec-
tron microscopy; Katia Georgopoulos, Lin Wu, Jeffrey Wu, and the Massachu-
setts General Hospital transgenic mouse core facility for assistance in the
generation of knockin mice lines; Kathryn Coser and the Massachusetts Gen-
eral Hospital Cancer Center DNA Microarray Core; and Emily McKillip, David
Wilson, and Seo-Kyoung Hwang for technical assistance. This work was sup-
ported by National Institutes of Health Grants to S.A.-T. (HG003616-01A1,
CA098402-06, and NS026084-18), J.K.L. (HL052233), and M.A.M. (5 P50
NS10828-32) and by Yale University School of Medicine (A.L.).
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