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Page 4 of 9 Finetti et al. Vessel Plus 2021;5:29 https://dx.doi.org/10.20517/2574-1209.2021.49

subsequent to the acquisition of a high proliferative capability. This clonal expansion is followed by a
[33]
massive recruitment of non-mutated endothelial cells that become part of the lesion [Figure 1] .
[34]
These first observations for CCM3 dependent cavernomas have been confirmed by Malinverno et al. . In
both mice and humans, the endothelial cells lining the lesion appear as a mosaic, formed by both mutated
and non-mutated endothelial cells. Only a very small part of this mosaic is made up of CCM3-/- endothelial
[34]
cells (about 15% of total endothelial cells of the lesion). Malinverno et al. report interesting data
demonstrating that CCM3-/- cells are able to recruit surrounding CCM3+/+ cells and to induce them to
undergo endothelial-mesenchymal transition (EndMT) through BMP6. Therefore, an important event in
the evolution of the cavernomas appears to be the attraction of CCM3+/+ cells by CCM3-/- cells, which, in
[34]
turn, strongly contribute to the growth of the malformation .

These new findings on CCM3 loss dependent vascular lesions have been confirmed also for CCM1/KRIT1.
In the paper by Rath et al. , immunohistochemical analysis of samples of a patient with double KRIT1
[30]
mutation revealed scattered immunopositivity and immunonegativity of CCM1 staining in endothelial of
caverns, while CCM2 and CCM3 were uniformly immunopositive, indicating that the second KRIT1
postzygotic mutation occurs only in a little number of endothelial cell present in the lesion.

In the light of the above observations, it is evident that CCM vascular lesions are not composed of a
homogenous layer of mutated endothelial cells, but may be defined as a mosaic of CCM mutated and non-
mutated endothelial cells [Figure 1]. These observations could also explain the difficulties in the
identification of the second postzygotic mutations, as the reduced number of mutated endothelial cells in
vascular lesions imposes the use of extremely sensitive techniques. Moreover, the mechanism by which wild
type endothelial cells become incorporated in the growing malformation is unknown even under
investigation. The identification of specific signal molecules able to promote the recruitment of wild type
endothelial cells into growing lesions may represent a challenge for the identification of new therapeutic
strategies.

NON-AUTONOMOUS EFFECTS OF CCM GENE LOSS
The hypothesis that CCM mutated cells become able to recruit non-mutated cells was initially formulated
for CCM3 by Louvi et al. . The authors showed that loss of CCM3 in astrocytes induces both cell
[35]
autonomous and non-autonomous effects. Ccm3-/- astrocytes showed increased proliferation and survival
through the activation of Akt signaling, but were also able to induce vascular lesion formation in mouse
[35]
models, with diffusely dilated and simplified vascular tree, similar to human cavernomas . Similarly,
[36]
Wang et al. , demonstrated in a mouse model that Ccm3 deletion in mural cells (pericytes and smooth
muscle cells) induces CCM lesions throughout the whole brain in mice. Vascular lesions are characterized
by a reduction in pericyte coverage and reduction of tight and adherens junctions of endothelial cells. In
addition, Ccm3 loss in brain pericytes reduced their migration and association with endothelial cells .
[36]
Finally, very recently, Lopez-Ramirez et al. proposed an interesting model in which the crosstalk between
[37]
astrocytes and endothelial cells drives vascular lesion formations. They showed that astrocytes are the major
source of VEGF during CCM development. The increased amount of VEGF production in astrocytes is
linked to increased hypoxia-inducible factor-1α (HIF-1α) activity in normoxic conditions promoted by the
augmented amount of nitric oxide (NO) produced by neighboring endothelial cells. The elevation of NO
production by endothelium is due to endothelial nitric oxide increasing after the elevation of the
transcription factors KLF2 and KLF4, previously implicated in the CCM pathogenesis. In addition, they
showed that the inhibition of cyclooxygenase-2 (COX-2), a HIF-1α regulated gene, ameliorates the
development of CCM lesions in animal models . These data confirm that CCM3 deficient stromal cells
[37]