Article

Developmental timing of CCM2 loss influences cerebral cavernous malformations in mice

Institut National de la Santé et de la Recherche Médicale, UMR-S 740, 75010 Paris, France.
Journal of Experimental Medicine (Impact Factor: 12.52). 08/2011; 208(9):1835-47. DOI: 10.1084/jem.20110571
Source: PubMed
ABSTRACT
Cerebral cavernous malformations (CCM) are vascular malformations of the central nervous system (CNS) that lead to cerebral hemorrhages. Familial CCM occurs as an autosomal dominant condition caused by loss-of-function mutations in one of the three CCM genes. Constitutive or tissue-specific ablation of any of the Ccm genes in mice previously established the crucial role of Ccm gene expression in endothelial cells for proper angiogenesis. However, embryonic lethality precluded the development of relevant CCM mouse models. Here, we show that endothelial-specific Ccm2 deletion at postnatal day 1 (P1) in mice results in vascular lesions mimicking human CCM lesions. Consistent with CCM1/3 involvement in the same human disease, deletion of Ccm1/3 at P1 in mice results in similar CCM lesions. The lesions are located in the cerebellum and the retina, two organs undergoing intense postnatal angiogenesis. Despite a pan-endothelial Ccm2 deletion, CCM lesions are restricted to the venous bed. Notably, the consequences of Ccm2 loss depend on the developmental timing of Ccm2 ablation. This work provides a highly penetrant and relevant CCM mouse model.

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J. Exp. Med. Vol. 208 No. 9 1835-1847
www.jem.org/cgi/doi/10.1084/jem.20110571
1835
Cerebral cavernous malformations (CCMs) le-
sions consist of densely packed vascular sinu-
soids lined by a thin endothelium with rare
subendothelial cells and no intervening paren-
chyma (Russel and Rubinstein, 1989; Clatterbuck
et al., 2001). Ultrastructural analysis of CCM
lesions showed defective tight junctions (TJ)
between endothelial cells (ECs). The lesions
form predominantly in the central nervous sys-
tem (CNS), but 5% of the patients also show
lesions in the retina (Labauge et al., 2007; Riant
et al., 2010).
The prevalence has been estimated to be up
to 0.5% in the general population. CCM oc-
curs as a sporadic (80% of patients) or a familial
disease (20% of patients), after an autosomal dom-
inant pattern of inheritance. Sporadic patients
most often have a single CCM lesion, whereas
patients aected by a hereditary form of the
disease have multiple lesions and show a pro-
gressive increase in lesion number over time.
Symptoms include headaches, seizures, and
focal neurological decits caused by cerebral
hemorrhages. The major risk for patients is the
recurrence of hemorrhages. Neurosurgery is
the only therapy oered today; however, it is
not always possible depending on the lesion
location within the CNS.
Familial CCM disease is caused by hetero-
zygous germline mutations in any of the three
CCM genes identied so far (CCM1/KRIT1,
CORRESPONDENCE
Elisabeth Tournier-Lasserve:
tournier-lasserve@
univ-paris-diderot.fr
Abbreviations used: AJ, adherens
junction; CCM, cerebral cav-
ernous malformation; CNS,
central nervous system; E, em-
bryonic day; EC, endothelial
cell; iCCM, inducible CCM
KO mice; LEF, lymphoid en-
hancer factor; P, postnatal day;
PCP, planar cell polarity; TCF,
T cell factor; TJ, tight junction;
Wnt, Wingless and Int.
N. Rudini and L. Maddaluno contributed equally to this paper.
Developmental timing of CCM2
loss inuences cerebral cavernous
malformations in mice
Gwénola Boulday,
1,2
Noemi Rudini,
3
Luigi Maddaluno,
3
Anne Blécon,
1,2
Minh Arnould,
1,2
Alain Gaudric,
4
Françoise Chapon,
5
Ralf H. Adams,
6
Elisabetta Dejana,
3,7
and Elisabeth Tournier-Lasserve
1,2,4
1
Institut National de la Santé et de la Recherche Médicale, UMR-S 740, 75010 Paris, France
2
Université Paris 7-Denis Diderot, Faculté de Médecine, Site Lariboisière, Paris, F-75010, France
3
Fondazione Italiana per la Ricerca sul Cancro Institute of Molecular Oncology (IFOM), 20139 Milano, Italy
4
AP-HP, Groupe Hospitalier Saint-Louis Lariboisiere-Fernand-Widal, Paris, F-75010, France
5
Department of Pathology, Centre Hospitalier Universitaire, Avenue Côte de Nacre, 14032 Caen, France
6
Max Planck Institute for Molecular Biomedicine, Faculty of Medicine, Department of Tissue Morphogenesis and University
of Münster, 48149 Münster, Germany
7
Department of Biomolecular Sciences and Biotechnologies, Milan University School of Sciences, 20133 Milan, Italy
Cerebral cavernous malformations (CCM) are vascular malformations of the central nervous
system (CNS) that lead to cerebral hemorrhages. Familial CCM occurs as an autosomal
dominant condition caused by loss-of-function mutations in one of the three
CCM
genes.
Constitutive or tissue-specic ablation of any of the
Ccm
genes in mice previously estab-
lished the crucial role of
Ccm
gene expression in endothelial cells for proper angiogenesis.
However, embryonic lethality precluded the development of relevant CCM mouse models.
Here, we show that endothelial-specic
Ccm2
deletion at postnatal day 1 (P1) in mice
results in vascular lesions mimicking human CCM lesions. Consistent with
CCM1/3
involve-
ment in the same human disease, deletion of
Ccm1/3
at P1 in mice results in similar CCM
lesions. The lesions are located in the cerebellum and the retina, two organs undergoing
intense postnatal angiogenesis. Despite a pan-endothelial
Ccm2
deletion, CCM lesions are
restricted to the venous bed. Notably, the consequences of
Ccm2
loss depend on the devel-
opmental timing of Ccm2 ablation. This work provides a highly penetrant and relevant CCM
mouse model.
© 2011 Boulday et al. This article is distributed under the terms of an Attribution
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The Journal of Experimental Medicine
Page 1
1836 New model of cerebral cavernous malformations | Boulday et al.
use to analyze the role of the CCM proteins during cerebral
angiogenesis and CCM pathophysiology.
In this study, to bypass embryonic lethality, we deleted the
Ccm2 gene in the mouse in an inducible, EC-specic manner.
Postnatal day 1 (P1) deletion of Ccm2 resulted in vascular
lesions that are strikingly similar to human CCM lesions in
the cerebellum and retina. Using the same strategy, P1 dele-
tion of Ccm1 or Ccm3 led to similar cerebellar and retinal
lesions. We showed that CCM2 lesion development is restricted
to the venous bed. We also demonstrated the existence of a
time window for the eects of Ccm2 deletion, related to a
developmental stage with intense angiogenesis. Here, we de-
scribe a relevant mouse model for CCM disease with com-
plete penetrance and fast development of the disease allowing
us to follow the lesion development.
RESULTS
Endothelial-specic
Ccm2
deletion at P1 results in vascular
malformations mimicking human CCM lesions in the CNS
To bypass the embryonic lethality previously encountered in
the Tie2Cre;Ccm2 KO mouse model (Boulday et al., 2009),
we crossed Ccm2 oxed mice with the endothelial-specic,
tamoxifen-inducible Cdh5(PAC)-CreERT2 mice (Wang
et al., 2010). Rosa26-Stop
-LacZ reporter mice were used
to monitor the tamoxifen-induced Cre/Lox recombination
(Soriano, 1999). As assessed by XGal staining, high recombi-
nation eciency was obtained after tamoxifen treatment
(Fig. S1, A–F). Unless otherwise mentioned, the genotype of
Ccm2-ablated mice (iCCM2 for inducible CCM2 KO) was
Cdh5(PAC)-CreERT2; Ccm2
/Del
; Rosa-Stop
-LacZ (see
Materials and methods for details of mouse breeding).
Animals were tamoxifen injected at P1 and were analyzed
at dierent time points, from P6 through 3 wk of age (Table I).
The estimated survival median for iCCM2 animals calculated
by the Kaplan-Meier test is 17 d (with a P < 0.001 of sig-
nicance for survival compared with controls; Fig. 1 A).
At P8, all iCCM2 animals were alive and grossly undistin-
guishable from controls. They did not show any signicant
dierence in weight (4.14 ± 0.6 g for iCCM2 versus 4.51 ±
0.7 g for controls). However, from P6 onward, we were able
to distinguish the iCCM2 animals upon dissection of the
brain (for 80% of the P6-P7 iCCM2 and 100% from P8
onward) and a clear aggravation of the vascular phenotype
over time was observed (Fig. S2 A). P1 deletion of Ccm2
resulted in the development of CCM lesions within the CNS
CCM2/MGC4607, and CCM3/PDCD10; Craig et al., 1998;
Laberge-le Couteulx et al., 1999; Liquori et al., 2003; Denier
et al., 2004; Bergametti et al., 2005). Recently, a “two-hit”
mechanism has been demonstrated in human ECs within
the CCM lesions (Gault et al., 2005; Akers et al., 2009;
Pagenstecher et al., 2009).
The three CCM genes encode scaold proteins without
any sequence homology. In the recent past, in vitro data ob-
tained in dierent cell types identied numerous CCM protein
partners that form complexes involved in various intracellular
signaling pathways (Faurobert and Albiges-Rizo, 2010). Con-
sistent with their involvement in CCM pathogenesis, the
three CCM proteins can interact in a cytosolic ternary com-
plex, where CCM2 acts as a bridge between CCM1 and
CCM3 (Hilder et al., 2007). However, the in vivo functions
of this ternary complex remain unclear.
When associated with the integrin 1-binding protein
ICAP-1, KRIT1 shuttles from the cytosol to the nucleus; its
function there remains to be determined. CCM2 interaction
with KRIT1 modulates KRIT1 tracking by sequestering
KRIT1 in the cytoplasm (Zawistowski et al., 2005). KRIT1
is a Rap1 eector and stabilizes the endothelial cell–cell junc-
tions (Glading et al., 2007). Recently, physical interaction be-
tween KRIT1 and CCM2 was proven to be required for
CCM protein localization at endothelial cell–cell junctions
(Stockton et al., 2010). KRIT1 has also been involved in vas-
cular lumenization and polarization of the endothelial tube in
vitro (Lampugnani et al., 2010).
Several studies have provided new insights into CCM
protein functions in vitro, including cytoskeletal remodeling,
cell–cell junction homeostasis, lumen formation, and polar-
ization. However, it remains to be claried which protein
complexes and signaling pathways are relevant in vivo, and
how the loss of function of any of the three CCM proteins
might explain the etiology of the CCM disease. To this aim,
relevant mouse models for the CCM disease are needed to
understand the molecular mechanisms involved in lesion
pathogenesis. Moreover, relevant mouse models reproducing
CCM lesions would allow development of preclinical thera-
peutic studies (Leblanc et al., 2009; Yadla et al., 2010).
Over the past few years, several groups reported constitu-
tive or tissue-specic KO mice for all three CCM genes.
Homozygous Ccm1-decient mice die at midgestation with
defects in the vascular structure and impaired arterial mor-
phogenesis (Whitehead et al., 2004). We and others have
shown that endothelial-specic ablation of Ccm2 severely
aects embryonic angiogenesis, leading to lethality at mid-
gestation (Boulday et al., 2009; Whitehead et al., 2009). In
contrast, neuronal-specic and smooth muscle cell–specic
deletion of Ccm2 does not aect the vascular development
(Whitehead et al., 2009). In a recent publication, He et al.
(2010) reported similar results using constitutive or tissue-
specic deletion of Ccm3 in mice. Together, those studies
demonstrated that endothelial expression of the CCM pro-
teins is crucial for proper angiogenesis. However, the embry-
onic lethality of these previous mouse models limited their
Table I. Number of iCCM2 analyzed after P1 deletion
Age of
analysis
Total numbers
of iCCM2
iCCM2 with cerebral CCM lesions
a
/
total number of iCCM2
P6-7 12 10/12
P8-10 30 30/30
P11-15 10 10/10
P16-19 4 4/4
a
Visible lesions upon dissection.
Page 2
JEM Vol. 208, No. 9
Ar ticle
1837
Ccm2 deletion in the mouse retina mimic the CCM retinal
lesions found in patients (Fig. 1 D, right).
We used the same gene targeting strategy to invalidate
endothelial Ccm1 or Ccm3 in mice. Tamoxifen-induced dele-
tion of Ccm1 or Ccm3 at P1 resulted in CCM malformations
in the CNS of all iCCM1 and iCCM3 animals analyzed (n = 4
in each group). The CCM1 and CCM3 lesions were compa-
rable to the lesions obtained after Ccm2 deletion in terms of
location (within the cerebellum and at the periphery of the
retina) and phenotype (Fig. S3).
We established a relevant mouse model for the human
CCM with a complete penetrance and a fast development of
CCM lesions from single to multiple hemorrhagic caverns
(Fig. 1 B and Fig. S2, C–F). Moreover, P1 deletion of any of
the three Ccm genes resulted in a similar phenotype according
to what is established for the human CCM disease.
Endothelial cell–cell junctions are altered in CCM2 KO cells
in vitro and in CCM2 lesions
To investigate whether part of the phenotype could be related
to altered endothelial cell–cell junctions, we analyzed the
expression and organization of junctional proteins in ECs
in vitro and in vivo in cerebellar vessels of
iCCM2 mice.
We abrogated CCM2 expression by
treating ECs derived from Ccm2
/
mice
with TAT-Cre recombinase (Fig. S4 A).
of all iCCM2 animals. Interestingly, the lesions were exclu-
sively located in the cerebellum (Fig. 1 B). Histology showed
cerebellar lesions composed of dilated vessels full of blood
cells (Fig. 1 B, bottom). In more severe cases, lesions form
multiple caverns, sometimes with evident signs of hemor-
rhage (Fig. 1 B, right). The mouse CCM lesions phenocopy
lesions found in human CCM patients (Fig. 1 D, left). At P8,
lesions of dierent severity were found in the same iCCM2
animal, from small vessel dilations to large caverns (Fig. S2, B–F).
From P9 onward, some iCCM2 animals were found dead in
their cage. When possible, analysis of these animals before
death revealed extensive cerebral hemorrhages, located mostly
around the multiple caverns composing CCM lesions but also
scattered all over the brain (Fig. 1 B, right; and not depicted).
In addition to CCM lesions, Ccm2 deletion at P1 in the
mouse induced dilation of vessels from the pial surface of the
cerebellum (Fig. 1 B, middle).
Because 5% of familial CCM patients show vascular
lesions in the retina, we analyzed the retinal vessels in iCCM2
mice. All iCCM2 animals showed CCM vascular malforma-
tions located at the periphery of the retinal vascular plexus in
both eyes (Fig. 1 C). Again, the vascular lesions obtained after
Figure 1. Endothelial
Ccm2
deletion at P1
results in CCM malformations mimicking the
human CCM lesions in the cerebellum and in the
retina. All animals were injected at P1 with 10 µl
tamoxifen (equivalent to 20 µg) and dissected at the
indicated time. Genotypes of inducible CCM2 KO
(iCCM2) and control animals were respectively
Cdh5(PAC)-CreERT2; Ccm2
Del/
and Cdh5(PAC)-Cre-
ERT2; Ccm2
+/
. (A) Kaplan-Meier survival curve from
the control group (blue line, n = 110) and the iCCM2
group (dotted red line, n = 56). Circles represent cen-
sored animals, which were sacriced for analysis.
(B) Control and iCCM2 mouse brains upon dissection
(top) and after H&E staining (bottom; n = 6 in each
group from 4 different litters, analyzed between P11
and P19). CCM malformations, located in the cerebel-
lum of iCCM2 animals, are composed by single or
multiple caverns (asterisks) with extensive hemor-
rhage (black arrows) around the juxtaposed vascular
cavities. Note the dilation of meningeal vessels in the
iCCM2 (yellow arrow). (C) Control and iCCM2 mouse
retinas at P13 upon dissection (left and middle) and
after isolectin-B4 staining (right, n = 7 in each group
from 4 different litters, analyzed between P11 and
P15). (D) Mouse lesions phenocopy human CCM
lesions. (left) Histology of the cerebral lesions in
mouse and human. (right) Mouse retina upon dissec-
tion and human retinal angiography Bars: 2 mm
(B, top); 500 µm (C and D, mouse retina); 100 µm
(B [bottom] and D [mouse cerebellum]).
Page 3
1838 New model of cerebral cavernous malformations | Boulday et al.
We then analyzed endothelial junction organization in vivo
in iCCM2 brain vasculature (Fig. 2). Staining of junctional
components supported data obtained in cultured ECs. Indeed,
claudin-5 or ZO.1 were correctly expressed in peri-lesion
vessels, but strongly reduced in abnormally dilated and hem-
orrhagic CCM lesions (Fig. 2 B). In addition, the AJ protein
VE-cadherin appeared to be disorganized and was not clearly
concentrated at intercellular endothelial cell–cell contacts
within the lesion, as compared with normal cerebral vessels in
iCCM2 animals (Fig. 2 C).
Overall, these data indicate that CCM2 is required for
proper cell–cell junction architecture in ECs in vitro and that
CCM2 deletion in vivo results in impaired EC junction
architecture in CCM2 lesions.
To investigate the mechanism of the strong reduction in
claudin-5 expression upon CCM2 ablation, we evaluated
the major regulators of claudin-5 expression, including the
Akt–FoxO1 signaling pathway (Brunet
et al., 1999; Burgering and Kops, 2002;
Zhang et al., 2002; Daly et al., 2004; Taddei
et al., 2008), the transcription factor Sex-
determining region Y-box-18 (SOX18), and
the ETS ternary complex ELK3 (Fontijn
et al., 2008). Phosphorylation of Akt was
enhanced in conuent CCM2 KO ECs
compared with control ECs, accompanied
by a strong phosphorylation of FoxO1
(Fig. S5 A). These data excluded Akt–FoxO1
pathway involvement in claudin-5 reduction
in CCM2-null ECs. In contrast, SOX18 ex-
pression was found to be reduced by 50% by
quantitative RT-PCR in CCM2 KO versus
control ECs, whereas ELK3 expression was
not modied (Fig. S5 B). This result supports
the idea that alteration in SOX18 expression
In the absence of CCM2, ECs lost correct organization of
both TJ and adherens junctions (AJ; Fig. S4, B–D). Expres-
sion of the TJ component claudin-5 was essentially absent
in CCM2 KO ECs and was undetectable at junctions.
ZO.1 also presented a discontinuous and weak staining at
cellcell contacts, whereas JAM-A and Afadin appeared
diuse on the cell membrane (Fig. S4 D). AJ components
such as VE-cadherin, -catenin, and -catenin were sig-
nicantly reduced by CCM2 deletion and presented a dot-
ted, faint, and irregular staining at junctions (Fig. S4 D and
not depicted). Conversely, plakoglobin, another member
of the catenin family, was strongly increased, whereas
PECAM1 was unchanged (Fig. S4 C). Thus, in CCM2-null
ECs, the overall composition and organization of junctions
is strongly altered. In addition, endothelial monolayer per-
meability in the absence of CCM2 was signicantly in-
creased (Fig. S4 E).
Figure 2.
Ccm
2 deletion alters AJ and TJ organi-
zation in CCM lesions. Analysis of cell–cell junc-
tions in CCM2 malformations on frozen sections of
iCCM2 brain. For all immunouorescence experi-
ments, cell nuclei are visualized with DAPI (blue).
Data are representative of 3 independent observa-
tions (n = 5 in each group, from 2 different litters).
(A) H&E staining (left) and confocal microscopy analy-
sis showing vessels stained using anti-PECAM1 (red,
right). (B) Co-staining of the vessels using PECAM1
staining (red) and the TJ components (green) using
claudin-5 (top) and ZO.1 (bottom). Claudin-5 and
ZO.1 are normally expressed in peri-lesion vessels
(arrowheads), whereas they are strongly down-
regulated in abnormally dilated and hemorrhagic
vessels of the lesion (dotted area). (C) VE-cadherin
staining (red) of the endothelium lining lesion and
peri-lesion vessels. (right) Magnication of the boxed
area. Pink arrows indicate VE-cadherin expressed
outside of the junctions. Bars: 200 µm (A); 100 µm
(B); 60 µm (C, top); 4 µm (C, bottom and right).
Page 4
JEM Vol. 208, No. 9
Ar ticle
1839
2006; Fruttiger, 2007). Within the rst week of life a vascular
plexus develops at the inner surface of the retina, from the
central retina toward the periphery. At this stage, the strict
could be one of the mechanisms responsible for the down-
regulation of claudin-5 in the absence of CCM2.
The Wnt–-catenin pathway is not involved in CCM2 lesion
development in vivo
-Catenin participates in the formation and stabilization of
cadherin-based adhesion by forming a connection to the
actin cytoskeleton (Dejana, 2010). -Catenin is also a key ele-
ment of the canonical Wnt (wingless and Int-1) signaling that
promotes the nuclear localization of -catenin by blocking
the -catenin destruction complex. Binding of -catenin to
cadherins can antagonize Wnt signaling by sequestering
-catenin at the membrane. Alteration of AJ promotes
-catenin nuclear translocation and the subsequent activation
of T cell factor (TCF)/lymphoid enhancer factor (LEF) tran-
scriptional complexes. KRIT1 is localized at the endothelial
cell–cell junction and can be co-precipitated with -catenin
(Glading et al., 2007). Depletion of endothelial KRIT1 in
vitro has been described to induce -catenin delocaliza-
tion from the membrane to the nucleus leading to in-
creased -catenin transcriptional activity (Glading and
Ginsberg, 2010).
Interestingly, in CCM2 KO ECs in vitro, we also observed
a delocalization of -catenin from the membrane to the
nucleus, suggesting -catenin activation (unpublished data).
However, TCF/LEF--catenin transcriptional activity, as de-
tected using the TOPFlash reporter, which includes six TCF-
binding sites, was not signicantly modied in CCM2-null
cells compared with controls (Fig. 3 A), suggesting that the
Wnt–-catenin pathway is not disregulated in CCM2-null
ECs in vitro.
To investigate whether the Wnt–-catenin signaling
pathway is involved in vivo in the CCM pathogenesis, we
assessed the -catenin transcriptional activity in the absence
of CCM2 in mice. Animals were crossed with the BAT-Gal
reporter mouse (see Materials and methods for mating de-
tails), which drives -galactosidase expression under the
control of multimerized TCF/LEF binding sites for acti-
vated nuclear -catenin. iCCM2; BAT-Gal mice were in-
jected at P1 with tamoxifen to ablate Ccm2. Consistent with
the previous study by Maretto et al. (2003), the -catenin
signaling was activated in the mouse cerebellum at P8
(Fig. 3, B–I). We found a similar activation of the -catenin
signaling in controls or iCCM2 cerebellum, mostly located
in the Purkinje cell layer, and in some ECs within the white
matter. In contrast, the endothelium lining the CCM mal-
formations did not show any evidence of -catenin signal-
ing pathway activation at P8, neither in the cerebellum nor
in the retina (Fig. 3, G–I; and not depicted), suggesting an
absence of Wnt–-catenin pathway involvement in iCCM2
lesions at the time of analysis.
CCM lesions specically affect the venous bed
and not the arterial compartment
Retinal vessels develop after birth in the mouse, after a very
well dened and organized pattern (Dorrell and Friedlander,
Figure 3.
Ccm2
deletion has no signicant effect on Wnt–-
catenin signaling pathway in CCM2 KO ECs in vitro and in iCCM2
lesions. (A) TCF/LEF--catenin transcriptional activity in CCM2 WT and
null ECs in vitro was determined by transfecting CCM2 WT and KO ECs
with the TOP-TK-Luc or the FOP-TK-Luc reporter constructs (containing
WT or mutant Tcf/Lef binding sites, respectively, and a basal TK promoter,
upstream a luciferase gene). Columns are means ± SD of triplicates from
a representative experiment out of three performed. (B-I) Animals were
bred with the BAT-Gal reporter mouse to assess -catenin activation (see
Materials and methods for breeding details). All animals were injected
with tamoxifen at P1. XGal staining, performed on control and iCCM2
cerebellum, is shown (n = 8 in each group, from 3 different litters). White
arrows show the CCM lesions in iCCM2 animals (in C, E, and G). In F–I,
H&E staining was performed on cerebellum sections, after XGal staining.
The box in G is magnied in I. H shows a CCM lesion composed of
multiple juxtaposed caverns. Gcl, granular cell layer; ml, molecular layer;
pcl, Purkinje cell layer; wm, white matter. Bars: 1 mm (B and C); 500 µm
(D and E); 100 µm (F and G); 50 µm (H and I).
Page 5
1840 New model of cerebral cavernous malformations | Boulday et al.
early stages to determine herein the natural history of the
CCM lesion over time. We followed lesion development in
the retina with an isolectin-B4 staining (Fig. 5). As early as
P7, the vascular plexus was thicker at the venous leading
edge of the retina in iCCM2 animals. Quantication of
the vascular coverage at the venous leading edge showed a
29% increase in iCCM2 retinas compared with controls
(Fig. 5 B; 0.56 ± 0.01 in controls versus 0.76 ± 0.01 in
iCCM2; P = 0.0004; n = 4). By P9, the main veins were
dilated in iCCM2 animals with abnormal capillaries at the
periphery of the vascular plexus. In 3-wk-old control mice,
the retinal vasculature was fully established, with 3 distinct
plexuses. In contrast, at the periphery of iCCM2 retinas,
it was not possible to distinguish dierent vascular plex-
uses. The vasculature from the lesion was composed by
bubblelike vascular structures packed together. A 2,000-kD
intracardiac FITC-Dextran injection confirmed that
those abnormal vascular structures were lumenized (un-
published data).
To investigate the mechanisms of the increase in diameter
of iCCM2 retinal vessels at P7, we assessed whether EC pro-
liferation was altered in the absence of CCM2. Using a phos-
pho-histone-3 staining, we could not detect any signicant
increase in EC proliferation in iCCM2 compared with con-
trols at P6 (Fig. 5 C). These data suggest that EC proliferation
is not the primary event leading to CCM malformations and
does not contribute to the early vascular phenotype induced
by Ccm2 deletion.
alternation between arteries (thin and straight with avascular
spaces surrounding arteries) and veins (larger and more sinu-
ous) can easily be distinguished using an isolectin-B4 staining
(Fig. 4 A). Analyzing retina from 1-wk-old P1-ablated iCCM2
animals, we observed that CCM malformations at the periph-
ery of the iCCM2 retinas were clearly restricted to veins and
the surrounding capillaries (Fig. 4 A).
In utero Ccm2 deletion led to vascular anomalies in the
cerebral hemispheres, aecting cerebral rhinal veins and the
surrounding capillaries (Fig. 4 B). In contrast, middle cerebral
arteries remained normal. To further conrm that the arterial
compartment is not aected by CCM lesion, we compared
vessels from mice mated with either the Rosa26-Stop
-LacZ
reporter mouse (to visualize all the vessels), or with the
artery-specic EphrinB2
tlacZ
reporter mouse (Fig. 4, A [right]
and C; see Materials and methods for breeding details). XGal
stainings showed that endothelium of the retinal and cerebral
CCM lesions did not express the arterial-specic EphrinB2
marker. In addition, P1 deletion also resulted in dilation of
vessels running along the cerebellar folia at the meningeal
surface corresponding to dorsal cerebellar veins (Fig. 1 B;
Nonaka et al., 2002).
Collectively, our results showed that Ccm2 deletion aects
the venous bed and leads to capillary-venous malformations.
Natural history of CCM lesions
A great advantage of animal models is the possibility to
explore the etiology of the vascular malformations at very
Figure 4. CCM lesions are capillary-
venous and do not affect the arterial
compartment. (A) Analysis at P9 or P12 of
the retinal vasculature from control or iCCM2
animals using vascular isolectin-B4 staining
(left and middle, n = 25 in each group ana-
lyzed between P8 and P10), after XGal stain-
ing (right, n = 4 in each group, from 2
different litters). Tamoxifen-induced Ccm2
deletion was performed at P1. Arteries (A) are
thin and normal in iCCM2 retinas, whereas
veins (V) are dilated in iCCM2 animals. Aster-
isks show CCM lesions developing at the pe-
riphery of the retina. (B) Lateral view of
cerebral hemispheres of control and iCCM2
embryos dissected at E19.5 (n = 8 in each
group, from 4 different litters). Ccm2 deletion
was performed at E14.5. Vascular anomalies
affect the caudal rhinal vein (crhv) and the capil-
laries surrounding in the iCCM2 animals. The box
in the middle panel is magnied in the image on
the right. (C) Analysis of the cerebellar vessels at
P10 and P12 after XGal staining on whole brain
(left) and after a H&E staining (right). Animals
were crossed with either the Rosa26-Stop
-LacZ
reporter mouse or the artery-specic Ephrin-
B2
tlacZ
reporter mouse. Results shown are repre-
sentative of at least four animals in each group,
from two different litters. Bars: 1 mm (A and B,
left and middle); 500 µm (B [right] C [left]); 100
µm (A, right); 50 µm (C, right).
Page 6
JEM Vol. 208, No. 9
Ar ticle
1841
Collectively, our data on Ccm2 deletion during embryo-
genesis, the postnatal period, and at 3 wk of age strongly sug-
gested that the eects of Ccm2 deletion on CCM lesion
development were restricted to key time points temporally
related to the angiogenic process.
DISCUSSION
In the present paper, we used an inducible, EC-specic dele-
tion of the Ccm2 gene, to bypass the embryonic lethality
encountered after constitutive or endothelial-specic Ccm2
ablation (Boulday et al., 2009; Whitehead et al., 2009).
The pathological consequences of
Ccm2
ablation
are restricted to key time points temporally related
to intense angiogenesis
Ccm2 deletion at P1, a time of intense angiogenesis in the
mouse retina and cerebellum (Yu et al., 1994; Plate, 1999;
Acker et al., 2001; Dorrell and Friedlander, 2006) resulted in
CCM lesions in both organs within a week after deletion. To
assess whether the eect of Ccm2 deletion is temporally cor-
related to the angiogenic process we compared deletion of
Ccm2 at P1 with deletion either at 3 wk of age, after establish-
ment of retinal and cerebellar vessels (Acker et al., 2001;
Dorrell and Friedlander, 2006), or during embryogenesis,
when cerebral angiogenesis is at an intensive stage (Fig. 6;
Yu et al., 1994; Plate, 1999). Tamoxifen-induced recombina-
tion eciency was conrmed for the dierent protocols
used (Fig. S1).
Ccm2 deletion in 3-wk-old animals did not lead to any
gross cerebrovascular phenotype, as observed on iCCM2
brains upon dissection at 2 mo of age (n = 4; Fig. 6 A, middle).
Histology performed on iCCM2 brains did not reveal any
vascular lesion or any sign of hemorrhage in the cerebral
hemispheres and cerebellum (unpublished data). Moreover,
isolectin-B4 stainings of retinal vessels showed similar vascu-
lature in iCCM2 and control animals (Fig. 6 B). These data
show that Ccm2 deletion at 3 wk did not lead to CCM in the
mouse, suggesting that endothelial Ccm2 is dispensable for
mature vessels.
We and others have reported that endothelial-specic
Ccm2 deletion resulted in early embryonic death around em-
bryonic day 10.5 (E10.5; Boulday et al., 2009; Whitehead
et al., 2009). In the present work, tamoxifen-induced endo-
thelial-specic Ccm2 deletion was performed at E14.5 to by-
pass the early embryonic lethality. At E19.5, iCCM2 mice
were distinguishable from their hemorrhagic skin (unpub-
lished data). Upon dissection, iCCM2 brains showed vascular
anomalies located on the cerebral hemispheres with irregular
and tortuous rhinal cerebral veins surrounded by abnormal
capillaries (Fig. 6 A, right; n = 8).
To get some insight into the tightness of the time-window
for Ccm2 deletion-mediated vascular eects, Ccm2 was ab-
lated at dierent postnatal time points. P4 inactivation
(n = 4) resulted within 10 d after induction, in a milder vas-
cular phenotype compared with P1 inactivation, which was
still clearly visible upon dissection (Fig. S6 B, to be com-
pared with Fig. S2 A). Histology of the brain showed single
isolated caverns located within the cerebellum, without sign
of hemorrhage (Fig. S6 E). Lesions were also observed at the
periphery of the retinal vasculature (Fig. S6 H). After P8-
(n = 5) and P15- (n = 2) Ccm2 ablation, no obvious vascular
phenotype was detected upon dissection in the cerebellum,
at P16 and P34, respectively (Fig. 6 D and not depicted). How-
ever, histology analysis revealed vessel dilations that might
precede CCM lesion development (Fig. S6 F). Isolectin-B4
stainings also showed vessel dilations at the periphery of the
P8-ablated iCCM2 retinas (Fig. S6 J).
Figure 5. Natural history of the CCM lesions. (A) Retinal CCM lesion
development from P7 to P16. The vasculature at the periphery of the
retina on controls or iCCM2 animals is shown after isolectin-B4 staining.
C, central retina; p, peripheral retina. Data are representative of at least
50 animals in each group, analyzed between P6 and P19. (B) Quantica-
tion of the vascular coverage at the venous leading edge of the plexus in
the retina at P7. Data are expressed as vascular area ± SEM (isolectin-
B4–positive area, relative to total retinal area analyzed; n = 4 in each
group, from 2 different litter; 6–8 elds analyzed per retina). (C) Quanti-
cation of the EC proliferation in control or iCCM2 retinas at P6, assessed
by total number of phospho-histone 3–positive ECs per retina ± SD
(n = 6 in each group, from 3 different litters). Bars, 100 µm.
Page 7
1842 New model of cerebral cavernous malformations | Boulday et al.
to increase the rate of somatic muta-
tion of CCM genes and obtain vascu-
lar lesions in heterozygous Ccm1
+/
or
Ccm2
+/
mice. On a tumor repres-
sor Trp53-null background, 30% of
the Ccm1
+/
mice developed lesions
(Plummer et al., 2004), but the fre-
quency of early onset malignancies
was a limitation of this model. With a
similar approach, 50% of the Ccm1
+/
mice with a mismatch repair complex
null background (Msh2
/
; Ccm1
+/
)
developed lesions (McDonald et al.,
2011). No lesion was obtained in the
Msh2
/
;Ccm2
+/
mice.
This sensitized mouse model, as
well as the model from Louvi et al.
(2011), has the advantage of progress-
ing slowly, with a relative normal lifetime for the animal. In
addition, according to what is thought to happen in human,
lesion development in the heterozygous McDonald’s model
occurs as a stochastic event throughout the brain (McDonald
et al., 2011). In contrast, in the model described herein, a very
ecient loss of both Ccm2 alleles is obtained in ECs, provid-
ing a more aggressive model. iCCM2 animals after P1-induction
show a rapid onset of the disease, with lesion development
restricted to some specic locations of the CNS, followed by
an early death caused at least in part by severe hemorrhages
within the CNS. However, our results using later postnatal
Ccm2 deletion (Fig. S6) suggested that a milder mouse model,
useful for long term studies, could be obtained that might
allow lesion development within the brain hemispheres. This
will be the subject for further analysis. Finally, it would be
unrealistic to expect one animal model to fully recapitulate a
human phenotype and we believe that the dierent CCM
mouse models available so far will be complementary for
mechanistic exploration.
In our study, AJ and TJ organization was strongly aected
in CCM2-decient ECs in vitro and in the CCM2 lesions in
vivo, consistent with the impaired TJs described in human
CCM lesions. CCM2 deletion caused complex changes in AJ
and TJ organization, which included down-regulation of
Early post-natal deletion of Ccm2 resulted in vascular lesions
strikingly recapitulating human CCM lesions in the brain as
well as in the retina, in 100% of Ccm2-deleted animals. In this
robust and relevant mouse model for the human CCM dis-
ease, we showed that CCM lesions aect only the venous bed
and that CCM development is restricted to key time periods
temporally related to intense angiogenesis.
In the past few years, dierent groups working in the eld
of CCM in vivo models concluded to a vascular, EC-specic,
autonomous function of the 3 Ccm genes (Hogan et al., 2008;
Boulday et al., 2009; Whitehead et al., 2009; He et al., 2010).
Recently, a paper challenged those results showing CCM-like
lesions after neuroglial-specic loss of Ccm3. In contrast to
what was previously published, Louvi et al. (2011) demon-
strated a cell autonomous eect of Ccm3 in astrocytes, result-
ing in increased cell proliferation and cell survival, as well as a
cell nonautonomous eect, resulting in a vascular phenotype.
This data suggest that Ccm genes play a role in vascular and
non vascular cells within the CNS, pointing out the impor-
tance of the communication between cells composing the
neurovascular unit, which may partly explain the CNS re-
striction of the CCM lesions.
Another approach to obtain a mouse model for human
CCM has been developed using genetic sensitizers, attempting
Figure 6. Timing of ablation determines
endothelial response to CCM2 loss. Control
and iCCM2 animals were injected with tamox-
ifen to delete Ccm2 at P1 (left, n = 25 in each
group, analyzed between P8 and P10), at
3 wk of age (middle, n = 4 in each group,
from 3 different litters) or at E14.5 during
gestation (right, n = 8 from 4 different litters).
(A) Control and iCCM2 brains upon dissection.
(B) Isolectin-B4 staining on control and
iCCM2 retinas. Note the CCM lesion in the
P1-induced animal (asterisks). V, vein. Bars:
2 mm (A, left and middle); 500 µm (A [right]
and B [left]); 100 µm (B, right).
Page 8
JEM Vol. 208, No. 9
Ar ticle
1843
Within the brain and the retina, CCM lesions aect only
the venous bed. This is consistent with what is observed in
human retinal CCM lesion when using retinal angiography.
The bubblelike vascular structures composing the CCM reti-
nal lesion are the last vessels to be lled up by the uorescent
dye, suggesting that lesions are composed by pocket-like cap-
illaries connected to the venous system. In zebrash, Ccm1 or
Ccm2 knockdown using morpholinos did not aect dorsal
aorta and intersomitic vessel development, but resulted in ab-
normal morphogenesis with major dilation of the posterior
cardinal vein and the caudal vein (Hogan et al., 2008). In our
study, mechanisms explaining the venous restriction of CCM2
lesions remain to be elucidated. The venous-specic eect of
CCM2 ablation cannot be explained by a dierence in the
timing of excision that would aect a vascular bed with a later
development, because veins and arteries of the supercial vas-
cular plexus develop concomitantly in the retina (Dorrell and
Friedlander, 2006; Fruttiger, 2007). We then excluded a dier-
ence in recombination eciency between veins and arteries.
As assessed by XGal staining on retinas or cerebral hemi-
spheres, P1-tamoxifen–induced recombination was clearly
aecting arteries and veins to the same extent (Fig. S1, A and
B). Another trivial explanation would be a venous restriction
of Ccm2 expression during late embryogenesis and the post-
natal period. In a previous work, we detected a moderate la-
beling for all three Ccm transcripts in the heart, arterial, and
venous large vessels by E14.5, decreasing at late embryogenic
stages (Petit et al., 2006). To further address this issue, we com-
pared CCM2 mRNA and protein expression in mesenteric
arteries and veins. No signicant dierence in Ccm2 gene ex-
pression level was observed in these two vascular beds at the
perinatal period (unpublished data). CCM2 protein expres-
sion was also conrmed in both types of vessels (unpublished
data). The venous specicity of CCM lesions could also
reect a dierent level of TJ component expression (i.e., clau-
din-5 expression) in veins versus arteries. However, claudin-5
expression, evaluated by quantitative RT-PCR was similar in
mesenteric arteries and veins (unpublished data). Thus, addi-
tional work is needed to clarify what diers between veins
and arteries that could explain the specic response of venous
EC to Ccm2 deletion.
The main characteristic feature of aected veins at early
stages of lesion development in retinas of the iCCM2 mice
was an increase in the size of the veins. To understand the
mechanisms of this venous dilation, we analyzed EC prolifer-
ation before lesion formation. We did not nd any enhance-
ment in EC proliferation, suggesting that proliferation is not
the primary event leading to lesions. This is consistent with
what was found by other groups in the mouse as well as in the
zebrash (Hogan et al., 2008; McDonald et al., 2011). In
iCCM2 cerebellum, the endothelium lining the already
formed CCM lesions (single or multicavernous) did not show
any increase in cell proliferation compared with endothelium
from controls, as assessed by stainings for the proliferation-
associated nuclear protein Ki67 at P8 and P14 (Fig. 2 J and
not depicted). Our results contrast with other data, showing
junctional components and alteration of their distribution at
intercellular contacts. Surprisingly, endothelial cell–cell TJs
were maintained in the Msh2
/
; Ccm1
+/
mouse model as
assessed by electronic microscopy (McDonald et al., 2011).
The apparent discordance between this model and our data
may come from the dierent approaches used by the two
groups. In our model, we cannot exclude that, even though
we observed a down-regulation and alteration of some AJ and
TJ components using immunostaining approaches, the TJ
ultrastructure could be preserved. Previous studies (Whitehead
et al., 2009; Stockton et al., 2010) showed that in CCM2-
decient ECs, cortical actin cytoskeleton was severely af-
fected. This eect required the association of CCM2 with
CCM1/Krit. Data presented here add to these observations
and show that deletion of CCM2 causes complex changes in
AJ and TJ organization, which include down-regulation of
junctional components and alteration of their distribution
at intercellular contacts. In a previous paper, we showed
(Lampugnani et al., 2010) that similar junctions’ alterations
could also be observed in CCM1-depleted ECs, further sup-
porting the idea of a physical and functional interaction
between CCM1 and CCM2. One of the most striking eects
of CCM2 depletion observed in our study was the strong re-
duction of claudin-5 expression, which is likely the cause of
altered TJ organization. This eect may explain, to a good ex-
tent, the defect in permeability control of CCM2 KO ECs in
vitro and in vivo (Fig. S4; Stockton et al., 2010). Furthermore,
as already mentioned, TJ are severely aected in the vascular
lesions of CCM patients. Genetic deletion of claudin-5 is
known to be associated to defects in blood–brain barrier (Morita
et al., 1999), which leads to death immediately after birth.
Another functional consequence of alterations in AJ or TJ
architecture is defective cell polarity. We previously reported
that Ccm1 silencing altered VE-cadherin and AJ organization
and inhibited the localization of the polarity complex at cell–
cell junctions (Lampugnani et al., 2010). As a consequence,
the polarized expression of apical (podocalyxin) and basal
(collagen IV) proteins was aected. In this study, although
junctions are altered in vivo in CCM2 lesions, apical and basal
proteins seem to be correctly distributed (Fig. S2 H). It is rea-
sonable that because CCM1, but not CCM2, also directly in-
teracts with integrins and modulates their functions, this
additional property may be required for cell polarity (Zovein
et al., 2010).
Our results clearly showed that despite pan-endothelial
Ccm2 ablation, CCM lesions did not aect all vascular beds.
CCM lesions developed only in the cerebellum and the retina
after P1 ablation. At the time of analysis, other highly vascular-
ized organs, such as the heart and lungs, did not show evidence
of CCM lesions upon dissection, even though Cre-mediated
recombination was conrmed in those organs (unpublished
data). Thus, our data clearly demonstrate that loss of Ccm2 is not
sucient to induce CCM lesions. In addition to the complete
endothelial absence of CCM2, additional factors, possibly spe-
cic for the neurovascular microenvironment, might be neces-
sary to cause the CCM disease.
Page 9
1844 New model of cerebral cavernous malformations | Boulday et al.
approaches to obtain mouse models for the CCM disease. All
these very recent complementary in vivo studies show simi-
larities but also dierences, most likely linked to distinct
methodological approaches, which will be useful to decipher
the mechanisms of CCM development.
In this study, we describe a relevant and robust mouse
model for CCM disease with a complete penetrance in the
CNS. We believe this model to be of importance in better
deciphering molecular mechanisms involved in the CCM
pathogenesis. Moreover, the rapid onset of the disease in the
iCCM2 mouse model makes it particularly suitable for thera-
peutic preclinical evaluation, especially for a fast rst screen-
ing of novel agents targeting lesion genesis. Indeed, prevention
of lesion development/progression/bleeding, or induction of
lesion regression are now the real challenge to pursue for the
CCM disease (Yadla et al., 2010).
Herein, analysis of the iCCM2 model suggests that the
loss of Ccm2 is required and sucient for the development of
CCM lesions but only in a restricted spatial and temporal
manner. We propose that, within an appropriate time window,
a pro-angiogenic stimulus in the neurovascular unit micro-
environment provides a permissive signal for venous EC from
the CNS to eventually form CCM lesions.
MATERIALS AND METHODS
In vivo tamoxifen-induced deletion. Tamoxifen (Sigma-Aldrich) was
diluted in sunower oil-10% ethanol at 10 mg/ml, and subsequent dilutions
were performed in sunower oil when necessary. For postnatal deletion, pups
were injected at P1 with a single intragastric injection of 20 µg tamoxifen.
Deletion at 3 wk of age was performed by repeated i.p. injections of 1 mg
tamoxifen for 4 consecutive days. For deletion during embryogenesis,
pregnant females were injected once i.p. with 1 mg tamoxifen at E14.5, and
a cesarean was performed at E19.5.
Mouse lines. The strategy used to target the Ccm2 gene in mice (Ccm2
oxed and Ccm2 deleted alleles) was previously described (Boulday et al.,
2009). Ccm1 and Ccm3 oxed mice were made by Taconic. The Cdh5(PAC)-
CreERT2 mouse line was previously reported (Wang et al., 2010). The
Rosa26-Stop
-LacZ (Soriano, 1999), EphrinB2
tlacZ
(Wang et al., 1998), and
BAT-Gal (Maretto et al., 2003) mice have been purchased from The Jackson
Laboratory. Mice were all bred on a C57BL/6 background.
To obtain the iCCM2 mice, the Cdh5(PAC)-CreERT2 mice were rst
bred with the Ccm2
+/Del
animals. The Cdh5(PAC)-CreERT2; Ccm2
+/Del
mice
were then crossed with Rosa26-Stop
-LacZ; Ccm2
/
animals. Thus, unless
otherwise mentioned, the genotype of iCCM2 animals and controls were as
follows: Cdh5(PAC)-CreERT2; Ccm2
/Del
; Rosa26-Stop
-LacZ and Cdh5(PAC)-
CreERT2; Ccm2
/+
; Rosa26-Stop
-LacZ.
EphrinB2
tlacZ
and BAT-Gal mice were bred with Ccm2
/
animals before
be crossed with Cdh5(PAC)-CreERT2; Ccm2
+/Del
mice. All procedures de-
scribed in this study were in full accordance with the Institutional Animal
Care and Use Commitee “Lariboisiere-Villemin” (Committee number 9,
Paris, France).
Cell culture. Mouse lung ECs were derived from lungs of 3-mo-old
Ccm2
/
mice (Boulday et al., 2009) and immortalized as previously de-
scribed (Dong et al., 1997; Balconi et al., 2000). Conditional deletion of
Ccm2 in vitro was obtained using TAT-Cre fusion protein which is known
to promote the nuclear translocation of Cre recombinase (Peitz et al.,
2002). As a control, cells were treated either with buer or with an inactive
form of TAT-Cre. Sparse ECs were washed with HyQ ADCF mAb me-
dium (Thermo Fisher Scientic) and treated with 100 µg/ml TAT-Cre for
an increase in proliferation of EC lining multiple mature
caverns as compared with single, early cavernous lesions
(McDonald et al., 2011). It is possible that the relatively short
median survival in our mouse model may be a limit for ana-
lyzing EC proliferation in mature, multicavernous CCM
lesions. Interestingly, loss of Ccm1 in zebrash resulted in im-
paired EC morphology rather than increase in EC prolifera-
tion, with a progressive spreading and thinning of the ECs
forming the dilated vessel (Hogan et al., 2008). We do hy-
pothesize that such a mechanism could explain the pheno-
type described in our CCM2 mouse model.
In this paper, we showed that the timing of Ccm2 deletion
(E14.5, P1, and 3 wk of age) denes the cerebral (or retinal)
EC response to CCM2 loss. We rst excluded dierences in
tamoxifen-induced recombination eciency that could ex-
plain the disparate temporal and spatial responses to Ccm2
deletion. XGal staining conrmed a high recombination
eciency at E14.5, P1, and 3 wk of age, in all the cerebral and
retinal vessels (Fig. S1, A–F). Mice induced at 3 wk, after vessel
development, did not develop CCM lesion in the CNS. In
contrast, mice induced at P1 showed CCM lesions in the cere-
bellum and the retina, whereas late in utero Ccm2 deletion
elicits vascular malformations in the cerebral hemispheres.
In those two situations, the location of CCM lesions in the
CNS corresponds to specic places undergoing intense angio-
genesis at the time of deletion (Plate, 1999; Acker et al., 2001;
Dorrell and Friedlander, 2006). Thus, our results comparing
the dierent timing of Ccm2 deletion strongly suggest that
angiogenesis might be the extra trigger leading to CCM
lesion development. Interestingly, in human CCM patients,
the number of lesions increases signicantly with age, particu-
larly after 50 yr old (Denier et al., 2006; Labauge et al., 2007).
In addition, it has been shown that angiogenesis can occur in
human adult brain in response to cerebral ischemia (Beck and
Plate, 2009). We speculate that an increase in CCM lesion
number over 50 yr old may be related to proangiogenic stimuli
that may be caused by hypoxic events that occur with aging.
The mechanisms of this restricted temporal CCM com-
petence are thus far unknown. In some aspects, it is reminis-
cent of the previously reported restricted temporal cystogenic
competence of renal epithelial cells. The autosomal dominant
polycystic kidney disease is characterized by a progressive in-
crease in renal tubular diameter followed by multiple cysts
formation. A key postnatal developmental switch has been in-
volved in this cystogenic process and has been related to ab-
normal planar cell polarity signaling (PCP; Fischer et al.,
2006; Piontek et al., 2007; Karner et al., 2009; Verdeguer
et al., 2010). PCP controls, through the coupling of cell divi-
sion and morphogenesis, the growth and the size of the nor-
mal renal tube. Interestingly, in the normal developing retinal
vasculature, orientation of mitosis along the vessel axis was
also reported (Zeng et al., 2007), suggesting that vessel growth
is determined by PCP.
While this manuscript was in revision, two other studies
were published (Chan et al., 2011; Cunningham et al., 2011)
that independently validated the use of inducible Ccm KO
Page 10
JEM Vol. 208, No. 9
Ar ticle
1845
Immunouorescence was analyzed using a Nikon Eclipse 80i micro-
scope or by confocal microscopy (TCS-SP2-AOBS; Leica).
Statistics. Student’s two-tailed nonpaired t test was used to determine sta-
tistical signicance for in vitro analysis. The signicance level was set at
P < 0.05. Kaplan-Meier test was used to determine survival curve of the
iCCM2 animals versus controls.
Online supplemental material. Fig. S1 shows the analysis of tamoxifen-
induced recombination. Fig. S2 shows additional analysis of CCM2 lesions in
the cerebellum. Fig. S3 shows the cerebellar and retinal CCM lesions ob-
tained after Ccm1 and Ccm3 ablation at P1. Fig. S4 shows the analysis of AJ
and TJ junctions in vitro in CCM2 KO ECs. Fig. S5 shows the analysis of
molecular regulators of claudin-5 expression in vitro in CCM2 KO ECs. Fig. S6
shows the analysis of impact of the postnatal timing for Ccm2 ablation on the
vascular phenotype severity. Online supplemental material is available at
http://www.jem.org/cgi/content/full/jem.20110571/DC1.
We deeply thank Eric Vicaut for the Kaplan-Meier analysis, Pierre Lacombe for FITC-
Dextran perfusions, Siham Mallah and Maëlle Coquemont for excellent technical
help, and Anne Joutel for very helpful discussions.
This work was supported by the Agence Nationale pour la recherche grant
ANR-07-MRAR-002-01 (to E. Tournier-Lasserve), the Leducq Fondation grant 07
CVD 02 Hemorrhagic Stroke (to E. Tournier-Lasserve and E. Dejana), Institut National
de la Santé et de la Recherche Médicale, and grants from the French association of
CCM patients. G. Boulday is a postdoctoral fellow supported by the Leducq
Foundation.
The authors have no conicting nancial interests.
Submitted: 23 March 2011
Accepted: 26 July 2011
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100 µM chloroquine for 60 min at 37°C (Liebner et al., 2008).
TOP/FOP assay. The assay was performed using a previously described
standard technique (Taddei et al., 2008). In brief, 6 × 10
5
CCM2 WT and
null cells were plated in 6-well plates to form 80% conuent cultures at
time of transfection. Cells were transfected 24 h after seeding using the
LipofectAMINE-2000 method (Invitrogen), in accordance with the manu-
facturers instructions. To normalize for transfection eciency, a pCMV--Gal
plasmid was co-transfected. 3 g of either TOP-TK-LUC or FOP-TK-LUC
(containing WT or mutant Tcf/Lef binding sites and a basal TK promoter,
upstream a luciferase gene, respectively) was used in combination with 1 µg
pCMV--Gal. Luciferase activity was assayed 48 h after transfection, using
the Enhanced Luciferase Assay kit (BD). TCF/LEF -catenin–mediated gene
transcription was dened by the ratio of TOP-TK-LUC/FOP-TK-LUC
luciferase activities, where the -Gal activity of the internal control reporter
pCMV--Gal was used to correct dierences in transfection eciency.
Western blot analysis. Western blot analysis was performed according to
standard protocols. In brief, conuent cells were washed with PBS and
lysated by boiling in a modied Laemmli sample buer (2% SDS, 20% glycerol,
and 125 mM Tris-HCl, pH 6.8). Lysates were incubated for 10 min at 95°C
to allow protein denaturation. The concentration of protein was determined
using a BCA Protein Assay kit (Thermo Fisher Scientic) according to the
manufacturer’s instructions. Equal amount of proteins were loaded on gel and
separated by SDS-PAGE, transferred to a Protran Nitrocellulose Hybridiza-
tion Transfer Membrane 0.2 µm pore size (Whatman), and blocked for 1 h
at room temperature in TBST (150 mM NaCl, 10 mM Tris-HCl, pH 7.4, and
0.05% Tween)-powdered milk. The membranes were incubated overnight at
4°C in primary antibodies diluted in TBST-5% BSA or in TBST-5% milk.
Next, they were incubated for 1 h at RT with horseradish peroxidase-linked
secondary antibodies (diluted in TBST-5% milk). Membranes were rinsed
3 times with TBST for 5 min each, and specic binding was detected by the
enhanced chemiluminescence system (GE Healthcare) using Hyperlm (GE
Healthcare). The molecular masses of proteins were estimated relative to the
electrophoretic mobility of the co-transferred, pre-stained protein marker
Broad Range (Cell Signaling Technology).
Histology, -galactosidase staining, and immunouorescence. Brains
were xed by immersion overnight in 4% paraformaldehyde, before
being paran-embedded. For histology analysis, hematoxylin and eosin
(H&E) staining was performed on every 10 sections (10 µm each) on half-
sagittal brain.
Whole-mount staining for -galactosidase activity was performed by
incubation overnight with XGal (1mg/ml solution) after 30 min xation in
cold 1% formaldehyde xation buer.
Immunouorescence on frozen sections was performed after xation in
aceton or cold methanol. Sections were counterstained with DAPI and
mounted in a uorescent mounting medium (Dako). The following antibodies
were used for immunohistochemistry and/or immunouorescence: rat anti-
PECAM (MEC13.3, BD); rat anti–VE-cadherin (BD); rabbit anti-ZO.1
(Zymed); rabbit anti-claudin-5 (from H. Wolburg, Institute of Pathology,
University of Tubingen, Tubingen, Germany); rabbit anti-phosphohistone H3
(Abcam); peroxidase-conjugated anti–rat (Jackson ImmunoResearch Labora-
tories); Alexa Fluor 594–conjugated anti–rat (Invitrogen); Alexa Fluor 488–
conjugated donkey anti–rabbit (Invitrogen); FITC-conjugated anti–rabbit
(Jackson ImmunoResearch Laboratories).
Immunouorescence on whole-mount retinas was performed as previ-
ously described (Pitulescu et al., 2010). Staining of retinal vessels was ob-
tained by incubations with biotin-conjugated isolectin-B4 (AbCys) and
Cy3-streptavidin (GE Healthcare). NIS-Elements imaging software (Nikon)
was used to quantify vascular coverage at the venous leading edge of the reti-
nal vasculature (vascular area relative to the total retinal area analyzed) in
control and iCCM2 retinas (n = 4 animals with similar weight in each group,
6–8 elds per retina).
Page 11
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  • Source
    • "CCM1 is a known Notch activator, and brain endothelial cells from iCCM1 mice show Notch inhibition and upregulation of BMP6 leading to enhanced transforming growth factor β (TGF-β) and bone morphogenetic signaling protein (BMP) signaling resulting in CCM lesions [39]. In addition, Boulday et al. established a relevant mouse model where endothelial cell-specific deletion of Ccm1, Ccm2, and Ccm3 at postonatal day 1 (P1) resulted in vascular lesions that are similar to human CCM lesions and were predominantly restricted to the venous bed of cerebellum and retina [3]. This study comes in contrast to the other reports where the Notch components are especially expressed in the arterial and capillary beds. "
    [Show abstract] [Hide abstract] ABSTRACT: Cerebral cavernous malformations (CCM) commonly known as cavernous hemangioma are associated with abnormally enlarged thin-walled blood vessels. As a result, these dilated capillaries are prone to leakage and result in hemorrhages. Clinically, such hemorrhages lead to severe headaches, focal neurological deficits, and epileptic seizures. CCM is caused by loss of function mutations in one of the three well-known CCM genes: Krev interaction trapped 1 (KRIT1), OSM, and programmed cell death 10 (PDCD10). Loss of CCM genes have been shown to be synergistically related to decreased Notch signaling and excessive angiogenesis. Despite recent evidences indicating that Notch signaling plays a pivotal role in regulating angiogenesis, the role of Notch in CCM development and progression is still not clear. Here, we provide an update literature review on the current knowledge of the structure of Notch receptor and its ligands, its relevance to angiogenesis and more precisely to CCM pathogenesis. In addition to reviewing the current literatures, this review will also focus on the cross talk between Delta-Notch and vascular endothelial growth factor (VEGF) signaling in angiogenesis and in CCM pathogenesis. Understanding the role of Notch signaling in CCM development and progression might help provide a better insight for novel anti-angiogenic therapies.
    Full-text · Article · Jan 2016 · Neurosurgical Review
    • "Analysis of the number/size of the CCM cavernae was performed using ImageJ software. Three different groups of caverns were defined according to their size (area in lm 2 ) (Boulday et al, 2011). For immunohistochemistry on human samples, 3-lm paraffin sections were stained with hematoxylin and eosin (H&E) to assess the histological features. "
    [Show abstract] [Hide abstract] ABSTRACT: Cerebral cavernous malformations (CCMs) are vascular malformations located within the central nervous system often resulting in cerebral hemorrhage. Pharmacological treatment is needed, since current therapy is limited to neurosurgery. Familial CCM is caused by loss-of-function mutations in any of Ccm1, Ccm2, and Ccm3 genes. CCM cavernomas are lined by endothelial cells (ECs) undergoing endothelial-to-mesenchymal transition (EndMT). This switch in phenotype is due to the activation of the transforming growth factor beta/bone morphogenetic protein (TGFβ/BMP) signaling. However, the mechanism linking Ccm gene inactivation and TGFβ/BMP-dependent EndMT remains undefined. Here, we report that Ccm1 ablation leads to the activation of a MEKK3-MEK5-ERK5-MEF2 signaling axis that induces a strong increase in Kruppel-like factor 4 (KLF4) in ECs in vivo. KLF4 transcriptional activity is responsible for the EndMT occurring in CCM1-null ECs. KLF4 promotes TGFβ/BMP signaling through the production of BMP6. Importantly, in endothelial-specific Ccm1 and Klf4 double knockout mice, we observe a strong reduction in the development of CCM and mouse mortality. Our data unveil KLF4 as a therapeutic target for CCM.
    No preview · Article · Nov 2015 · EMBO Molecular Medicine
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    • "To study the contribution of autophagy to CCM pathogenesis, we investigated whether KRIT1 down-regulation would lead to the impairment of autophagy in endothelial cell lines. Endothelial-specific KRIT1 knockout (KO) in mice produced lesions that were identical to the CCM malformations observed in humans (Boulday et al, 2011; Maddaluno et al, 2013). We used KRIT1-KO lung endothelial cells derived from KRIT1 fl/fl mice treated with Tat-Cre recombinase (Maddaluno et al, 2013). "
    [Show abstract] [Hide abstract] ABSTRACT: Cerebral cavernous malformation (CCM) is a major cerebrovascular disease affecting approximately 0.3-0.5% of the population and is characterized by enlarged and leaky capillaries that predispose to seizures, focal neurological deficits, and fatal intracerebral hemorrhages. Cerebral cavernous malformation is a genetic disease that may arise sporadically or be inherited as an autosomal dominant condition with incomplete penetrance and variable expressivity. Causative loss-of-function mutations have been identified in three genes, KRIT1 (CCM1), CCM2 (MGC4607), and PDCD10 (CCM3), which occur in both sporadic and familial forms. Autophagy is a bulk degradation process that maintains intracellular homeostasis and that plays essential quality control functions within the cell. Indeed, several studies have identified the association between dysregulated autophagy and different human diseases. Here, we show that the ablation of the KRIT1 gene strongly suppresses autophagy, leading to the aberrant accumulation of the autophagy adaptor p62/SQSTM1, defective quality control systems, and increased intracellular stress. KRIT1 loss-of-function activates the mTOR-ULK1 pathway, which is a master regulator of autophagy, and treatment with mTOR inhibitors rescues some of the mole-cular and cellular phenotypes associated with CCM. Insufficient autophagy is also evident in CCM2-silenced human endothelial cells and in both cells and tissues from an endothelial-specific CCM3-knockout mouse model, as well as in human CCM lesions. Furthermore, defective autophagy is highly correlated to endothelial-to-mesenchymal transition, a crucial event that contributes to CCM progression. Taken together, our data point to a key role for defective autophagy in CCM disease pathogenesis, thus providing a novel framework for the development of new pharmacological strategies to prevent or reverse adverse clinical outcomes of CCM lesions.
    Full-text · Article · Sep 2015 · EMBO Molecular Medicine
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