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Padarti et al. Vessel Plus 2018;2:21 I http://dx.doi.org/10.20517/2574-1209.2018.34 Page 9 of 23

in FAK and Pyk2 . Through fluorescence images, it was determined that paxillin and CCM3 were co-
[63]
localized to the plasma membrane at the leading edge . The functionality of this binding is still unknown,
[102]
one hypothesis is that the formation of some CCM3-GCKIII complexes is under the control of paxillin
phosphorylation. Therefore, paxillin may be sequestering CCM3 from activity .
[103]

CELLULAR SIGNAL TRANSDUCTION
CCM1 plays a role in Notch signaling
Cells with increased CCM1 activity show overexpression of HEY1 and DLL4, two major players in notch
signaling. Notch signaling increases PI3K/AKT signal pathway and activated AKT leads to suppression
of ERK1/2 by dephosphorylation. CCM1 deficient cells and CCM lesions show increased phosphorylation
of ERK1/2 . AKT phosphorylation is also important in regulating the expression of SOD2, which is an
[104]
important free radical scavenger in the cell. SOD2 is upregulated with increases in reactive oxygen species
through AKT phosphorylation. In the absence of CCM1, AKT phosphorylation is decreased [72,105] , leading
to decreased expression of SOD2, and therefore increasing oxidative damage in the cell . CCM1 is also an
[106]
inducer of SOD2 through interaction of ND1. ND1 is an important actin stabilization protein that binds to
CCM1. This interaction increases the expression of SOD2. Therefore, CCM1 can prevent oxidative damage
and cell death through complex induction of SOD2 .
[107]
CCM1 involved in KLF4/KLF2 signaling pathways
Both in vivo studies with Ccm1 knockout mice and in vitro studies with CCM1 silencing in human brain
EC (hCMEC) showed elevation of KLF4 nuclear signal. KLF4 has been reported to play an important role
in EC in biogenesis of veins and angiogenesis in general. Combined silencing of both KLF4 and CCM1
significantly decreases the disease mortality (75% reduction of mouse mortality) and modest improvement
of vascular lesions (reduced vascular density in retina). The prototypical lesions in CCM lack mesenchymal
intervening tissue, which is due to increased proliferation and dysfunctional migration, both of which are
mediated with KLF4 inhibition. In this signaling pathway, CCM1 binds and sequesters MEKK3, which in
turn activates MEK5, which subsequently activates ERK5 [108,109] . ERK5 is a known inducer of KLF4 in EC
cells [110-112] . KLF4 is a transcription factor which activates BMP6 and decreases SMAD1 phosphorylation. It
has been shown that SMAD is activated in CCM deficient condition and leads to active BMP6 and TGF-β.
These two downstream proteins mitigate the histological manifestation of CCM, i.e., lack of intervening
parenchyma . KLF2 is another transcription factor that is induced by a similar signaling cascade . KLF2
[114]
[113]
is responsible for the cardiac manifestations and increased angiogenesis seen in CCM [115,116] . KLF4/KLF2 are
transcription factors that suppress expression of thrombospondin1 (TSP1) which functions as an angiogenic
inhibitor . Loss of TSP1 was found to exacerbate CCM phenotype in Ccm1 deficient mice. Abnormalities of
[117]
cell-cell junction are found to be the initial manifestation of CCM1. It was found that decreases in Claudin-5
and ZO-1 levels were the first to be observed before changes in VE-cadherin levels. The perturbed expression
of these cell junction proteins can be rescued in Ccm1 knockout mice with exogenous TSP1 (3TSR). 3TSR
was found to decrease both VEGR2 phosphorylation leading to decreased angiogenesis and increased TGF-β
activation. Furthermore, this treatment decreases the lesion burden in mice. Therefore, there is a possibility
to utilize KLF4 inhibitors, ERK5 inhibitors, or exogenous TSP1 for potential therapeutic applications in
CCM in the near future .
[118]

CCM1 is a key player in integrin signaling
β1-integrin signaling is an important regulator in many cellular functions such as cellular migration and
adhesion [15,24,25,57] . These functions are especially important for EC cells. ICAP1α is a repressor of β1-integrin
signaling . Our previous results indicate that CCM1 binds to ICAP1α and modulates ICAP1α and β1-integrin
[46]
interaction. Depletion of CCM1 and ICAP1α synergistically inhibits extracellular signal-regulated kinase/
mitogen-activated protein (ERK/MAP) kinase pathway activation on cell survival [15,23-25,57,72] . We hypothesized
that CCM1 regulated recruitment of ICAP1α to the cell membrane in proximity to focal adhesions, which