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Padarti et al. Vessel Plus 2018;2:21 I http://dx.doi.org/10.20517/2574-1209.2018.34 Page 11 of 23

overexpression of vasodilators during angiogenesis. The phenotype of hyperangiogenesis in CCM3 deficient
condition can be rescued by overexpression of DLL4. Abnormal Notch signaling leads to many downstream
effects. Notch signaling is also reported to be involved in the regulation of expression of VEGF receptors,
to modulate vascular bed architecture and angiogenesis [39,130] . In fact, CCM3 deficient cells increase
[129]
the expression levels of VEGF which affects cell survival through ERK1/2 activity . ERK1/2 kinase was
[131]
reported to be upregulated in CCM3 deficient lesions, which can be reversed when DLL4 function is rescued
through induction of recombinant DLL4. Therefore, CCM3-mediated notch signaling also affects ERK1/2
and VEGF functions leading to abnormalities in EC cells. The finding that CCM3 deficient phenotype can
be rescued through DLL4 overexpression creates a promising venue for future pharmacotherapy .
[126]

TLR4 signaling in CCM pathogenesis
It was found that induction of gram negative bacterial abscesses in Ccm1 and Ccm2 deficient mice significantly
increased the phenotypic severity of CCM lesions. The effect was increased in mice with hematogenous
infections of gram-negative bacteria. In fact, exposure to just lipopolysaccharide (LPS) was sufficient for
significant lesion formation in the mice. It is well known that LPS response is mediated by TLR4 pathway
[132]
and that MEKK3 deficiency terminates the signal . MEKK3-KLF2/4 pathway has already been implicated
[133]
in CCM lesion formation , which is further validated by decreased expression of KLF2/KLF4 with an
[90]
LPS injection, suggesting the existence of TLR4-MEKK3-KLF2/4 pathway. Furthermore, heterozygous TLR4
mutants had a significantly decreased CCM lesion formation and homozygous mutants had a complete
resolution of CCM lesions. This suggests that TLR4 signaling drives CCM lesions development. In fact,
genetic polymorphisms in TLR4 (rs10759930) and CD14 (rs778587) (a TLR4 co-receptor) that result in
increased expression of respective proteins do result in increased CCM lesions. These findings were further
supported by lack of CCM lesion formation in mice that were surgically removed as fetuses and grown in
sterile conditions. Therefore, exogenous stimulation of TLR4 may be involved in CCM lesion formation. Two
additional experiments were also performed to prove post-natal suppression of lesion can be achieved: (1)
TLR4 antagonists can decrease lesion severity in mice through decreased TLR4 signaling; (2) the course of
antibiotics also decreases lesion severity. These antibiotics altered the nature of the microbiome in the mice
for decreased TLR4 stimulation. This report provides a new avenue for potential CCM pharmacotherapy .
[134]

CELLULAR FUNCTIONS AND BIOGENESIS
Angiogenesis
CCM lesions are hallmarked by abnormally increased EC proliferation. There are many proposed mechanisms
for angiogenesis implicated in CCM. ICAP1α is involved in NOTCH signaling. Therefore, loss of CCM1 or
ICAP1α results in increased angiogenesis . CCM3 is also reported to be involved in NOTCH signaling
[104]
resulting in increased angiogenesis . CCM proteins also modulate MAP kinase activity which in turn
[126]
modulates angiogenesis . It is also reported that CCM3 increases VEGF receptor concentrations, thereby
[89]
resulting in increased EC proliferation . However, there is still controversy of the mechanistic pathways
[135]
for control of angiogenesis by the CCM proteins [23,136] .

Microvascular integrity
CCM lesions are not only hallmarked by the presence of abnormal EC proliferation and migration but
also increase the leakage predisposing the lesions to hemorrhage. There are several explanations for this
phenomenon. ANKS1B is a novel PTB domain containing protein that binds to the third NPXY motif in
CCM1. No change of EC cell proliferation, migration or sprouting was observed in ANKS1B-deficient EC,
suggesting that ANKS1B-deficiency did not affect the CCM1 activity or Notch signaling. However, these EC
cells had decreased transendothelial resistance (TER), which cannot be rescued by ROCK inhibitors. This
observation indicates that ANKS1B regulated EC cell adhesion without involving RhoA signaling. Further,
increased TER was achieved by overexpression of ANKS1B in CCM1 depletion, indicating that CCM1 is not
involved in ANKS1B signaling either . Another explanation is that the increased vascular permeability
[137]