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Page 12 of 23 Padarti et al. Vessel Plus 2018;2:21 I http://dx.doi.org/10.20517/2574-1209.2018.34

is due to stress fiber formation in CCM lesions. CCM proteins are responsible for inhibiting Rho kinase.
However, unopposed activation of Rho kinase results in increased phosphorylation of myosin light chain,
[58]
which was also seen in CCM lesions . Another theory is the dysfunction of RAP1 and CCM1. CCM1
acts as a scaffold for RAP1 and is important for cell junction protein β-catenin and VE-Cadherin, thereby
stabilizing the cell membrane. VE-Cadherin is a part of the adherens junction . Therefore, dysfunction
[138]
of VE-Cadherin affects cell contacts. β-catenin is a nuclear factor. Dysfunction of CCM proteins results in
increased translocation of β-catenin in the nucleus that lead to EC proliferation .
[139]
The vascular permeability seen in Ccm1 knockout mice can be rescued by SOD2 and catalase infusions with
antibody-mediated targeting to the endothelium. TNF-α was able to induce vascular permeability in the
arterioles in these rescued mice. However, TNF-α is unable to induce vascular permeability in Ccm1 null
mice. It was also shown that in the absence of CCM1, TNF-α was unable to generate ROS. This suggests
that TNF-α function involves CCM1. Yet, CCM lesions have shown to have elevated ROS. This is a result
of increased activity of NADPH oxidases (NOX). Both in vitro and in vivo CCM lesions showed significant
up-regulation of NOX4. NOX4 is not only a source of ROS, but also an enhancer for downstream activation
of NF-κB. This can be rescued by treatment of NOX4 inhibitors. Furthermore, broad spectrum NF-κB
inhibitor (i.e., N-(E)-p-coumaroyl-3-hydroxyanthranilic acid, YAv1) inhibits NF-κB activation due to NOX4
[140]
or TNF-α, which can rescue the defective endothelial barrier seen in CCM . These inhibitors could
potentially be utilized for CCM pharmacotherapy in the future.

CCM1 protects cells from oxidative damage
All three CCM proteins follow a knudsonian pattern of inheritance. Therefore, a second mutation is
necessary for development of lesions and reactive oxidative species are a source of DNA mutations. Initially,
Ccm mouse models were made with Ccm1 and Ccm2 heterozygous mutant mice. However, these mice
never showed any CCM lesion phenotype, so another mutation, Msh2, was developed into these mice. Msh2
is a DNA damage-repair protein that decreases DNA mutations. Only mice with heterozygous Ccm1 and
homozygous Msh2 deletion showed considerable vascular CCM lesions . Ccm1 is a regulator of FoxO1,
[101]
through an unknown mechanism. This transcription factor induces the transcription of Sod2 and Sirt1, two
important anti-oxidants in the cell . It was also shown that CCM1 can regulate Rho GTPase, by interacting
[106]
with ND1-L in the presence of oxidative stress . Another potential pathway modulated by CCM1 to limit
[107]
oxidative stress is JNK/c-Jun signaling. In CCM1-null cells, there is an overexpression of C-Jun which can be
reversed by reintroduction of CCM1. Therefore, CCM1 protects the cells from downstream oxidative stress
of C-Jun redox pathways . The cells lacking CCM1 also have increased activity of COX-2, a mediator of
[141]
[142]
inflammatory pathways in the cells . This is consistent with the in vivo data that Ccm1 knockout mice had
hyper-exaggerated response to inflammatory agents . Therefore, it can be concluded that inflammation
[143]
and oxidative stress are involved in CCM lesion formation . CCM1 provides protection against oxidative
[144]
stress in the cell by utilizing anti-inflammatory and anti-oxidant pathways .
[106]
CCM is associated with increased ROS that mediate cellular damage. The cell usually responds through
up-regulation of anti-oxidant enzymes. CCM1 depletion induced lesions show increased levels of Nrf2
transcription factor and Glo1 enzyme, both having important anti-oxidant functions in the cell. This results
in a paradoxical increase in cell death due to protective mechanisms. However, chronically activated anti-
oxidant mechanisms result in impairment of regular redox reactions in the cell. Many other vascular diseases
have overactivation of Nrf2 [145,146] . It was shown that chronic Nrf2 activation as seen in CCM is paradoxically
associated with increased ROS production. This, in accordance with other studies, shows that the anti-
oxidative effects of Nrf2 are only seen at certain concentration levels . Loss of function (LOF) of CCM1
[147]
results in increased JNK signaling, which has been shown to lead to increased Nrf2 activity [148,149] . JNK
inhibitors resulted in decreased Nrf2 activation and restored ability of autophagy. Impaired autophagy is
seen in CCM lesions and rescue of this phenotype suggest that it is mediated by JNK signal pathway. Similar