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Padarti et al. Vessel Plus 2018;2:21 I http://dx.doi.org/10.20517/2574-1209.2018.34 Page 13 of 23

to Nrf2, supraphysiological concentrations of Glo1 result in increased sensitivity to oxidative stressors [150,151] .
These cells also have decreased levels of heat shock protein (HSP), Hsp70 and Hsp27, which have a protective
role in the cell by increasing cellular capacity to handle stressors. The sequential effects by Glo1 compound
to increase sensitivity to stressors and decrease tolerance, result in greater propensity towards intrinsic cell
death in CCM1 deficient condition .
[152]

Appropriate valvulogenesis is predicated on CCM1-CCM2 complex
The fluid stress on the EC cells is paramount for appropriate differentiation of the heart. It is shown that
abnormalities in blood flow through alteration of KLF2a/b activity affect heg1. Heg1 is localized to areas of
myocardium with increase fluid forces in zebrafish . It was found the increased Heg1 expression stabilized
[153]
Ccm1 in these cells. Therefore, overexpression of Heg1 resulted in increased Ccm1 leading to decreased
Klf2a activity. This decreased Klf2a activity desensitizes the cells to the fluid forces resulting in appropriate
valvulogenesis. In the absence of Ccm1, the endocardial cushions do not develop into functional valves.
Induced expression of Ccm1 to endocardial cushions in Ccm1 deficient zebrafish resulted in development
of valves. These cells were shown to increase Notch signaling and decrease Klf2a activity with the targeted
expression of Ccm1. Similar valvular defects were also seen with the inhibition of Notch activity .
[154]
Therefore, CCM proteins are involved in appropriate valve development .
[155]
CCM2 and CCM3 function coordinately in gonadogenesis
Ccm2 and Ccm3 gene expressions are upregulated in adult mouse testis and ovaries, correlating with
CCM2 and CCM3 protein expression, suggesting the involvement of Ccm2 and Ccm3 in the regulation of
gonadogenesis. The expression pattern of CCM2 changes through embryogenesis. In the prenatal testis stage,
CCM2 is mainly expressed in the interstitial cells of Leydig with little expression in gonocytes. Throughout
gonad maturation, CCM2 begins to be expressed in spermatocytes, followed by the expression of CCM3.
In ovaries, CCM2 is found in the oocyte nucleus at birth. Overtime this expression is decreased while the
expression of CCM2 is increased in adult granulosa cells. The CCM2 in granulosa cells is expressed solely
in the cytoplasm based on the spatiotemporally differential expression patterns of CCM2 and CCM3 in
the testis and ovaries; it is plausible that CCM2 and CCM3 proteins may have different physiological roles
during testicular and ovarian development . Homozygous Ccm3 mutants in a C. elegans model rendered
[156]
them sterile. These Ccm3 mutant worms had multinucleated germ cells that showed hypoproliferation,
which may be caused by altered expression of Rab-11. Ccm3 promotes endocytic recycling by interacting
with Rab-11. Defective endocytic recycling could result in decreased expression of Glp-1, a mediator of
Notch signaling, and Rme-2, a mediator of protein endocytosis. Ccm3 deficient germ cells have defective
late-stages of cytokinesis leading to multinucleate giant cells. Polarity of C. elegans is modulated by non-
[157]
muscle myosin , while non-muscle myosin distribution is regulated by Ccm3. Ccm3-null embryos have
aberrant expression of Par-6 and Par-2, both of which are polarity proteins. Therefore, it can be concluded
that embryonic polarity is mediated by Ccm3 .
[158]

CCM2 plays a role in the cardiac phenotype seen in CCM
As we described before, CCM2 binds to MEKK3 through the HH domain, leading to increased expression in
KLF2, KLF4, ADAMTS4, and ADAMTS5 . These expression changes can be detected in the earliest stage
[115]
of CCM lesions. The increased KLF4 and KLF2 were not only found in CCM lesions but also blood vessels in
the cerebellum in Ccm1 knockout mice. These increased expressions were also reported in both sporadic and
familial forms of human CCMs. Also, their early presence suggests that KLF4/KLF2 may be involved in the
formation of lesions. MEKK3 is a MAP3 kinase that controls KLF2/KLF4 activity which is especially critical
in cardiogenesis. Ccm1 null mutant mouse model is embryonic lethal, but Map3k3 haploinsufficiency is able
to rescue this lethality. To determine the temporality of the Rho activation vs. MEKK3 activation, CCM1
depleted EC cells treated with hydroxyfasudil (a Rho inhibitor) was unable to normalize the levels of KLF4/
KLF2 while normalized KLF4/KLF2 levels in CCM1 null cells resulted in normal level of Rho activation,