2. Cerebral cavernous malformations (CCMs) consist of clusters of
enlarged endothelial channels (‘caverns’) that are arranged back-to-
back to form densely packed sinusoids with little or no intervening
brain parenchyma. These lesions lack smooth muscle and elastic
tissue, so the vessel walls are thin, leaky and lack sub-endothelial
support and an intact basal lamina. Ultrastructural analysis has
revealed ruptures in the luminal endothelium (probably due to
damaged intercellular junctions), endothelial detachment from the
basal lamina and decreased numbers of pericytes.
3. Cerebral Cavernous Malformations (CCMs) are common
vascular abnormalities, affecting the blood vessels of the
central nervous system in up to 0.5% of the population. They
are characterized by dilated, thin-walled vessels and can lead
to strokes, seizures and death due to recurrent brain
hemorrhages.
Cavernomas constitute 10% to 15% of all vascular
malformations, only 20% to %30 of affected persons develop
symptoms.
Adult presentation is mostly seen at the 4th and 5th decades.
Due to unknown reasons very high prevalances are reported in
Mexicans and the familial form of the disease is also more
frequent in Mexicans.
4.
5.
6. Familial CCM accounts for only 20% of cases but tends to be
more severe than sporadic CCM, with patients exhibiting multiple
lesions and increased hemorrhage rates. Familial CCM is
associated with a heterozygous germline loss-of-function
mutation in CCM1/KRIT1, CCM2/Malcavernin or CCM3/PDCD10. In
lesions with identified mutations, CCM1/KRIT1 is mutated in 65%
of cases, CCM2 in 19%, and CCM3/PDCD10 in 16%.
All three proteins can be found in the same complex within the
cell. However, CCM3/PDCD10 might also act separately from
CCM1/KRIT1 and CCM2/Malcavernin, as its mutation often results
in a more severe form of the disease.
7. Familiar Cerebral Cavernous Malformation is caused by loss-of-
function mutations in one of these three genes involved in blood
vessels formation: CCM1 (also known as KRIT1), CCM2 (MGC4607,
OSM, Malcavernin) and CCM3 (PDCD10, TFAR15, mapped to
chromosome 3q25.2-q27 ).
8. CCM1/KRIT1 is the largest of the CCM proteins. It contains an N-
terminal Nudix domain followed by a stretch of three NPxY/F motifs,
a predicted ankyrin-repeat domain and a C-terminal FERM (band 4.1,
ezrin, radixin, moesin) domain. No catalytic activity has been
reported for CCM1/KRIT1, and it is thought to signal through its
many binding partners (see below). CCM1/KRIT1 is ubiquitously
expressed in early embryogenesis with pronounced endothelial
expression in large vessels.
CCM1/KRIT1 is observed in many different cellular compartments
and is actively shuttled between the cytoplasm and the nucleus. A
polybasic sequence within a CCM1/KRIT1 Nudix domain loop is
responsible for the targeting of CCM1/KRIT1 to microtubules,
although the mode of interaction is not yet completely understood.
CCM1/KRIT1 also localizes to endothelial cell boundaries or cell–cell
junctions through its FERM domain.
9. Rap1 binding
inhibits the binding
of CCM1/KRIT1 to
microtubules,
thereby enabling
the CCM1/KRIT1
and the
stabilization of
cell–cell junctions
10. FERM domain of CCM1 can also bind to the membrane anchor
protein heart of glass 1 (HEG1), a protein essential for CCM1/KRIT1
junction localization. In zebrafish studies, as CCM1/KRIT1 mutants
that are unable to bind either Rap1 or HEG1 do not rescue the KRIT1-
null (santa) phenotype, which is associated with defects in
cardiovascular development.
11. Another important binding
partner of CCM1/KRIT1 is integrin
cytoplasmic domain associated
protein-1 (ICAP1), a
phosphotyrosine binding (PTB)-
domain-containing protein that
negatively regulates β1 integrin
activation. In endothelial cells,
CCM1/KRIT1 also appears to
stabilize the ICAP1 protein, so
CCM1/KRIT1 loss leads to
decreased ICAP1 levels and
consequently increased β1
integrin activation.
12.
13.
14. CCM1/KRIT1 overexpression leads to increased
expression of HEY1 and DLL4 (indicative of Notch
activation). and, conversely, silencing of CCM1/KRIT1
diminishes Notch signaling.
Loss of CCM1/KRIT1 also reduces the expression of the
reactive oxygen species (ROS)-scavenging enzyme superoxide
dismutase SOD2 with consequent increases in the steady state
levels of ROS and AKT phosphorylation.
15. Endothelial-to-mesenchymal transition
(EndMT) has been described in different
pathologies, and it is defined as the
acquisition of mesenchymal- and stem-cell-
like characteristics by the endothelium.
Endothelial-specific disruption of
the Ccm1 gene in mice induces EndMT,
which contributes to the development of
vascular malformations. EndMT in CCM1-
ablated endothelial cells is mediated by the
upregulation of endogenous BMP6 that, in
turn, activates the transforming growth
factor-β (TGF-β) and bone morphogenetic
protein (BMP) signalling pathway. Inhibitors
of the TGF-β and BMP pathway prevent
EndMT both in vitro and in vivo and reduce
the number and size of vascular lesions in
CCM1-deficient mice.
16.
17. CCM1/KRIT1 is thought to harbor intramolecular binding sites, i.e.
its N-terminus can interact with its FERM domain. It is therefore
possible that CCM1/KRIT1, like other FERM proteins, adopts both
open and closed conformations through a ‘head-to-tail’ interaction.
The head–tail interaction probably occurs though the recognition of
the CCM1/KRIT1 NPxY/F motifs by the FERM domain, although the
specificity of this interaction is still unclear.
Changes in the conformation of CCM1/KRIT1 are thought to
regulate its localization; for example, microtubule binding is
associated with a presumed ‘closed’ conformation and ICAP1
binding is associated with a presumed ‘open’ conformation.
18.
19. All three CCM genes are important in endothelial cell biology and vascular
development. However, not all CCM gene products seem to perform the
same functions. While CCM1 and CCM2 can regulate the cytoskeleton by
small G proteins, the importance of CCM3 in this regulation is not clear.
Moreover, CCM1 and CCM2 form a complex with the endothelial specific
orphan receptor HEG1, but CCM3 can only be identified in that complex
when overexpressed.
22. CCM2 is a scaffolding protein with no
enzymatic activity and an expression
pattern very similar to that of CCM1
/KRIT1, including in arterial
endothelial cells of multiple tissues.
It acts as the hub of the CCM
complex by simultaneously binding
both CCM1/KRIT1 and CCM3/
PDCD10, in addition to a number of
other signaling proteins. CCM2
contains a predicted PTB domain at
its N-terminus and has recently been
shown to contain a helical domain at
its C-terminus termed the harmonin-
homology domain (HHD).
23. CCM2 is found throughout the cell and can shuttle in and out of the
nucleus, probably through its interaction with CCM1/KRIT1.
However, CCM2 binding has also been implicated in sequestration of
the CCM1/KRIT1–ICAP1 complex in the cytosol. CCM2 localization to
endothelial cell–cell junctions is lost following the loss of
CCM1/KRIT1 localization to cell-cell junctions, suggesting that it is
targeted there by CCM1/KRIT1.
The leaky vasculature in CCM lesions is explained by their weak and
disordered cell–cell junctions. The importance of CCM1/KRIT1 in
cell–cell junctions is underscored by its association with the
junctional proteins VE-cadherin, α-catenin, β-catenin, AF6 (afadin,
also known as MLLT4) and p120-catenin.
Loss of CCM1/KRIT1 reduces β-catenin and VE-cadherin at cell–cell
junctions, leading to increased nuclear β-catenin and upregulation
of its transcriptional targets. Activation of Rap1 (which stabilizes
CCM1/KRIT1 in junctions) inhibits β-catenin transcription in a
CCM1/KRIT1-dependent manner.
24.
25. Brain hemorrhage in
inducibly deleted,
adult CCM2 mutant
mice. Gross view and
coronal sections are
shown. Brain lesions
are detected in the
cerebrum and
cerebellum of 7–8
months-old mice
26. Brain hemorrhage in
inducibly deleted,
adult CCM2 mutant
mice. Gross view
and coronal sections
are shown. Brain
lesions are detected
in the cerebrum and
cerebellum of 7–8
months-old mice.
E,G normal, F, H-
CCM2 knockout
27. Timing of ablation determines
endothelial response to CCM2 loss.
Control and iCCM2 animals were
injected with tamoxifen 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.
28. Fibrous matrix deposits (blue) identified by Masson’s trichrome
staining with fibrous tissue surrounding vascular channels (arrows)
and in surrounding gliotic brain. (G and H) Endothelial staining for
CD34 (G) or CD31 (H) is positive in the cells lining the channels. (I
and J) Elastin staining shows that vascular channels lack elastic
laminae (arrows). The fibrous matrix surrounding channels includes
laminin (K and L) and collagen IV (M and N).
Pathologic analysis of mouse
and human CCM2-
associated CCM. (A and B)
H&E staining revealing back-
to-back vascular channels
(arrows) and hemosiderin
pigment (arrowhead) in
surrounding
tissues. (C and D) Iron (blue)
detected by Prussian blue
stain highlights hemosiderin
deposits in macrophages and
surrounding brain tissue. (E
and F)
29. CCM2 deletion alters AJ and TJ organization
in CCM lesions. Analysis of cell–cell junctions
in CCM2 malformations on frozen sections of
iCCM2 brain. For all immunofluorescence
experiments, cell nuclei are visualized with
DAPI (blue). Data are representative of 3
independent observations (n = 5 in each group,
from 2 different litters). (A) H&E staining (left)
and confocal microscopy analysis 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) Magnification 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).
30. CCM3 is the least common -- and most aggressive --
familial form. Less than 2 percent of CCM cases in
the U.S. are known to carry the CCM3 mutation.
CCM3/PDCD10 is ubiquitously expressed and
contains an N-terminal dimerization domain and a C-
terminal focal adhesion targeting-homology (FATH)
domain. It binds a variety of proteins including CCM2,
the GCKIII serine/threonine kinases, paxillin (through
its FAT-H domain), FAP-1/PTPN13, protocadherin-c,
VEGFR, UNC13D and striatin.
Using morpholino gene knockdown technology in transgenic zebrafish
embryos expressing fluorescent proteins in their vasculature, an
excellent model was developed. Inhibition of CCM3 causes heart and
circulation defects distinct from those seen in CCM1 and CCM2
mutants, and leads to a striking dilation and mis-patterning of cranial
vessels reminiscent of the human disease.
31. Zebrafish CCM3 model presents an excellent
tool to study the development of CCMs in real
time. The vascular lesions develop quickly
(over the course of 72 hours) in a transparent
embryo, where the vasculature can be readily
imaged. Future research on zebrafish CCM3
morphants will allow the imaging and
mechanistic study of how endothelial
dysfunction first arises, and how the disease
is precipitated.
32. CCM3 binds to VEGF receptor VEGFR2 and to be important for its
signal transduction, and to bind to the focal adhesion protein paxillin
when overexpressed, two functions that have not been reported for
CCM1 or CCM2. Further, CCM3 interacts with all three members of
the GCKIII proteins, a family of protein kinases involved in the
response to cellular stress, Golgi biogenesis, and cytoskeletal
regulation. Importantly, it is this interaction, rather than its binding to
CCM1 and CCM2, what may be important for its role in endothelial
cell biology.
The GCKIII proteins are a group of three serine-threonine kinases
(Mst3/STK24, Mst4/MASK, and SOK1/YSK1/STK25) that belong to the
wider family of Ste20 kinases. GCKIII proteins have been related to
several important cellular processes. All three of them are activated
by oxidative stress, and while Mst3 and SOK1/STK25 are
proapoptotic, Mst4 has a prosurvival function
33. Endogenous CCM3 can interact with all three endogenous GCKIII
proteins (Mst3, Mst4, and SOK1), and have also shown that at least
part of CCM3 is on the cis face of the Golgi apparatus, and that it
influences Golgi morphology and cell polarity through GCKIII-
dependent phosphorylation of the adaptor protein 14-3-3ξ.
CCM3, but not CCM2, defects can be effectively rescued upon over-
expression of stk25b, a GCKIII kinase previously shown to interact
with CCM3. This suggests that Stk25b activity is downstream of
CCM3 in the cranial vasculature. In addition, morpholino
knockdown of the GCKIII gene stk25b results in heart and
vasculature defects similar to those seen in CCM3 morphants.
Finally, additional loss of CCM3 in CCM2 mutants leads to a
synergistic increase in cranial vessel dilation.
Cells depleted of CCM3/PDCD10 are impaired in repositioning both
the Golgi complex and the centrosome towards the leading edge,
which impairs cell migration.
34. Knockdown of CCM3 causes gross
enlargement and mis-patterning of
cranial vessels reminiscent of the
human disease pathology. b) CCM2
mutants display normal cranial
vasculature. c) Slight knockdown
of CCM3 in CCM2 mutants lead to
very severe cranial vascular defects.
d) This data, combined with stk25b
rescue of CCM3a/b morphants (but
not CCM2 mutants) suggests a model
where CCM1/2 and CCM3/STK25
signaling make up two distinct arms
of the CCM pathway that helps
regulate the development and
stabilization of the cranial
vasculature.
35. Cavernous malformations result from
LOH of either CCM2 or CCM3/PDCD10.
(A)Cavernous malformation (arrow)
shown in an H&E-stained section of
brain cerebrum from a mouse with
induced endothelial knockout
of CCM2.
(C) Cavernous malformations (arrows)
and a less complex telangiectasia
(arrowhead) shown in an H&E-stained
section of brain cerebrum from a mouse
with induced endothelial knockout
of CCM3/PDCD10.
36. A) Endothelial cells line the dilated
vascular channels (arrows ind.
endothel. nuclei). (B and C) IHC of
the endothelial lining (PECAM) of
two lesions (D) NADPH-diaphorase
stain(NOS activity) L, blood-
containing lumen of dilated vessel
(v) (E) H&E of a human lesion
resected from a patient with CCM.
Multiple caverns lined with a single
layer of endothelium are shown.
(F–J) TEM Pics (F, 7-mo-old; G–J,
10-mo-old mice). Erythrocytes (er)
in the luminal (L) and abluminal
sides of the dilated vessel. a, axon
in neighboring parench. (G) Tight
junctions (arrows) between
adjacent endothel. cells (e) lining
the lesion. c, connect. tissue; BM,
basement memb. (H) Intraluminal
thrombocytes (t) within lesion.
(I and J) Cell memb. (arrowheads)
of astrocytic end-foot processes (f)
abutting a dilated yet lesion-free
vessel (I) or the vascular lesion
proper (J) in the same brain.
Loss of cerebral cavernous malformation 3 (CCM3)
in neuroglia leads to CCM and vascular pathology
37. (A) Dilated vascular channels are lined by endothelial cells (arrowheads) with associated basal lamina (arrows). (B)
Occasional channels have segments with a multilayered appearance (arrows indicate lamellae of endothelium with
basal laminae). Tight junctions appear intact (arrowhead). (C) Focal areas of endothelial attenuation are observed
(arrow) without apparent gaps or disruption of tight junctions (arrowhead indicates a junctional complex). (D)
Channels are separated by loose connective tissue composed mostly of collagen (arrows). (E) Foci of mononuclear
inflammatory cells are present (arrows). (F) Hemosiderin-laden macrophages (arrow) are among the inflammatory
cells observed. Images are representative of 5 lesions from 3 CCM3/PDCD10 mice. Scale bars: 4 μm.
38. Vascular phenotypes in CCM3 neural mutants. (A and E) Cortical vessels visualized by in situ
hybridization for collagen 4a1. Vessels originating from the pial surface (red asterisks) and
invading the cortex seem disorganized in Gfap-CCM3 mutants (E) compared with controls (A).
(B, C, F, and G) Cerebral vasculature visualized after intracardiac administration of (sulfo-NHS)-
biotin. In Gfap-CCM3 mutants, the vessels are dilated (F), and the vascular tree is overall less
complex (G) compared with controls (B and C).
39. CCM3/PDCD10 is required in the
endothelium for venous integrity. (A–H)
H&E staining of developmental time
course of Pdcd10 endothelial knockout.
Pdcd10flox/+;Tie2-Cre is shown in A–D.
Pdcd10flox/–;Tie2-Cre is shown in E–H.
Close-up images of the cardinal vein at
E12.5 are shown in D and H. Asterisks
denote the cardinal veins. Circles denote
the external jugular veins. (I and J)
Echocardiography of hearts from
Pdcd10flox/– (I) and Pdcd10flox/–;Tie2-
Cre (J) mice at E11.5. Top panels show
M-mode images of hearts contracting
over time. Bottom panels show
waveforms corresponding to blood flow
across the atrioventricular valves. s,
systole; d, diastole; e, early filling; a,
atrial contraction; R, valvular
regurgitation.
40. The first indication that RhoA dysregulation might contribute to CCM
pathology came from the observation of increased stress fiber
formation (a sign of activated RhoA) after knockdown of any of the
CCM proteins.
Consistent with this, activated (GTP-bound) RhoA is increased in
CCM1/KRIT1, CCM2- or CCM3/PDCD10-deficient endothelial cells.
One of the primary effectors of activated RhoA is the
serine/threonine kinase ROCK, which increases actomyosin
contractility by phosphorylating and inhibiting the myosin light
chain (MLC) phosphatase.
Knockdown of CCM1/KRIT1, CCM2 or CCM3/PDCD10 increases the
amount of phosphorylated MLC, whereas treatment with ROCK-
inhibitors reverses this increase and the stress fiber accumulation.
41. How CCM proteins influence RhoA has still not been fully elucidated,
but a recent report implicates β1 integrin signaling. In addition,
CCM2 appears to direct the degradation of RhoA through
ubiquitination.
Cells that lack CCM1/KRIT1, CCM2 or CCM3/PDCD10 are defective in
migration, invasion, three dimensional tube formation and
maintenance of a monolayer permeability barrier. Each of these
functions can be rescued by ROCK inhibition. ROCK inhibitors also
rescue LPS-induced vascular leak in CCM1/KRIT1- and CCM2-
deficient mice.
42.
43. In animal models, CCM1/KRIT1, CCM2 and CCM3/PDCD10 are
essential for cardiovascular development. Loss of either KRIT1 or
CCM3/PDCD10 leads to an induction of angiogenesis by impaired
Delta–Notch signaling, and CCM3/PDCD10 might be essential for
venous endothelial cell differentiation.
Additionally, CCM1/KRIT1 deficiency disrupts the junctional
localization of the TIAM–PAR3–PKCf polarity complex, impairing
directed migration and vascular lumen formation. CCM3/PDCD10
is also important for endothelial cell polarization in directed
migration through its effects on Golgi positioning, further linking
CCM3/PDCD10 with vascular development.
44. Fasudil, a Rho kinase inhibitor, is able to
reduce development and hemorrhagic
rates of CCM lesions in a mouse model
of CCM-1 disease compared with placebo
controls. Lesions in treated animals were
smaller and less likely associated with
inflammation and endothelial
proliferation and exhibited decreased
expression of Rho kinase activation
biomarkers.
45. Fasudil hydrochloride is a
potent Rho-kinase inhibitor
and vasodilator. Since it was
discovered, it has been used
for the treatment of cerebral
vasospasm, which is often
due to subarachnoid
hemorrhage, as well as to
improve the cognitive decline
seen in stroke victims.
Actively being used in China
and Japan and is under
consideration by FDA.
46. Statin drugs may be used for treating CCM through the inhibition of
Rho GTPases. There is evidence that the Rho GTPase pathway can
be directly activated by ROS. Besides inhibition of Rho GTPases, the
serum cholesterol-lowering drug statin exerts powerful intracellular
antioxidant activities in endothelial cells, including the inhibition of
superoxide production and the improvement of ROS scavenging.
Antioxidant compounds may be safer than statins thanks to their low
side effects and thus may be used for treating CCM disease.
In particular, CCM1regulates the expression of the enzyme SOD2 and of its
transcription factor FoxO1 by protecting cells fromoxidative stress; therefore, a
loss of CCM1 leads to a significant increase in intracellular ROS levels and to
oxydative stress.
47. Sorafenib can ameliorate loss of CCM1-induced excessive
microvascular growth, by reducing the microvessel density
to levels of normal wild-type endothelial cells; in this way,
it could be a potential therapeutic approach also for
humans.
Sorafenib inhibits VEGFR, PDGFR and RAF kinases.
Approved for advanced renal, hepatocellular and thyroid
cancer.
